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VETERINARY GYNAECOLOGY
VGO-411 Credit Hours: 2+1
Instructors:

Dr. Ravi Dutt, Assistant Prof.

Dr. Sonu Kumari, Assistant Prof.

GENERAL TERMS AND BRIEF HISTORY

Vety. Gynaecology: study of patho-physiology of female reproduction in animals

Theriogenology: branch of veterinary medicine concerned with reproduction, including

the physiology and pathology of male and female reproductive systems of animals and the clinical
practice of veterinary obstetrics, gynaecology, and Andrology.

The term Theriogenology is derived from the ancient Greek words “Therio” meaning animal,
“gen” meaning creation/generation, and “ology” meaning study of. Theriogenologists are
veterinarians with advanced training in animal reproduction and obstetrics.

Andrology: study of patho-physiology of male reproduction.

Vety. Obstetrics: branch of veterinary medicine which deals with care and management of dam
during pregnancy; before, during and after parturition.
Reproduction: a complex process that involves many psychological and physio-chemical events
in genesis of a new individual. Reproduction has 3 purposes: species existence, food and genetic
improvement.
de Graaf(1672):described ovarian follicles
Johan Ham & Antonie van Leeuwenhoek (1677): described spermatozoa.

Embryology of Female Reproductive System

The fetal genital system consists of two undifferentiated gonads, two pair of ducts,
urogenital sinus genital tubercle and vestibular fold. This system develops from genital ridges
which are present on the dorsal side of abdomen of fetus. This system can develop either into male
or female embryo and this is known as embryonic bisexuality. In both the male and female during
embryonic period, 2 pairs of genital ducts are formed that enter the cloaca, the mesonephric/
wolffian tubules and ducts and the paramesonephric/ mullerian ducts.In male, Paramesonephric/
Mullerian duct degenerate and mesonephric/ wolffian duct persists. In female, Mesonephric/
Wolffian duct denenerate and paramesonephric/ mullerian duct persists
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Embryological Adult Female Adult Male

structure
Gonads Ovary Testis
Mesentery Mesovarium Mesorchium
Gubernaculum Round ligament of uterus, proper Ligamentum testis
ligament of ovary
Paramesonephric duct/ Oviduct, Uterus, Cervix, -Regresses
Mullerian duct vagina(cranial portion) Appendix testis and
Uterus masculinus
(remanants)
Mesonephric/ Wolffian -Regresses Efferent ducts,
duct Epoophoron, Paroophoron, Parovarian Epididymis, Vas-
cyst, Gartner‟s duct (remanants) deferens
Urogenital sinus Caudal part of vagina and urethra Accessory sex glands
and pelvic urethra
Genital tubercle Clitoris Penis
Genital folds Vestibule Penile urethra
Genital swellings Vulvar lips Scrotum

Female Reproductive System

Consists of ovaries, oviducts, uterus, vagina and vulva

1. OVARIES: paired, primary reproductive organs which produce female gamete and hormones.
Attached to broad ligament by a fold of peritonium k/a Mesovarium
 The ovary consists of medulla and cortex and surrounded by surface epithelium. Cortex
contains follicles, corpus haemorrhagicum, CL and atretic follicles.
 Main functions: production of oocyte; production of estrogen, progesterone, relaxin and
inhibin.
 Blood supply- highest at luteal phase, decreases with luteal regression and reaches nadir
just before ovulation.
 Ovary consists of Medulla (inner) and Cortex(outer)
Medulla: rich in connective tissue, blood vessels and nerves
Cortex : contains follicles
 Inflammation: oophoritis

Shapes of ovaries in different species:

Cow/buffalo : oval/almond shaped

Mare : Bean/kidney shaped (Ovulation fossa present)
Sheep/Goat : Almond shaped
Bitch/Sow : Mulberry like due to extensive follicular development
Camel : Oval
 Active ovary is slightly larger than inactive. In ruminants right ovary is more active,
attached with mesorchium dorsally and laterally; ventrally attached with utero-ovarian lig.
These ligaments form a pocket like structure, known as Ovarian bursa
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 Mare is left ovulator (60% ovulations from left ovary). Ovulation occurs at ovulation
fossa.
 Bitch: ovarian bursa contains fat
 Blood supply to ovary is by Utero-ovarian artery
 Follicle can be :
1) Primary: are surrounded by single layer of granulosa cells. There are about 150000
primary follicles in fetal ovaries. Group of primary follicles is known as Egg
nesting
2) Secondary: are surrounded by ≥2 layers of granulosa cells
3) Tertiary follicle: When fluid filled cavity (Antrum)is formed in the follicle.
4) Graffian follicle: Mature follicle containing antrum/fluid filled structure. It is
surrounded by fibrous layer. It appears like a blister.
Outer layer of graffian follicle-Theca externa; Inner layer of graffian follicle -Theca
interna(secretes estrogen hormone)
Granulosa cell at the base of ovum form cumulous oophorous on which oocyte rest.

Oocyte‟s outer layer: Corona radiata; Oocyte‟s Inner most layer: Zona pellucida

Cross section of bovine ovary

Diagram of a Graffian Follicle

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Atretic follicle: In each cycle many follicles develop, one grows and ovulates; rest undergo
degeneration. This degenerated follicle is k/a Atretic follicle.

Liquor folliculi /Follicular fluid: contains many hormones end enzymes which help in growth
and maturation of oocyte e.g. E2, P4(at ovulation),androgens, progestins, albumin, globulin, IgM,
FSH, LH/prolactin, cholesterol, PGE, PGF2α, IgG(predominant; conc. increases as follicle enlarge
to preovulatory size), IgA (in amount second to IgG), glucose, fructose, heparin hyaluronic acid,
glysine, aspartate, threonine, alanine, Na, K, Zn, Cu, Ca, P, S, Mg,Cl etc. Follicular fluid helps in
maturation of oocyte.

Size of Graffian follicle:

Cattle: 12-20mm
Buffalo: 10-12mm
Mare: 35-40mm

Corpus Haemorrhagicum(CH): Ovulation-Follicle collapse-cavity formation-Bleeding in

cavity-Cavity filled with blood-CH. CH is freshly formed CL, brick red in colour, soft on
palpation and starts projecting out of ovarian surface.
Corpus luteum (CL): CH is replaced by CL. It is rich in blood supply. Contains 2 types of cells:
a) Large lutein cells: originate from granulosa cells b) Small lutein cells: originate from theca
cells. Max size of CL (20mm) is attained on day 15-16 of estrous cycle. After day 16CL regress
and P4 level drop simultaneously. CL is yellow coloured (Yellow body) in cow and mare, whereas
grey coloured in bitch and sow.CL is nipple like in appearance.

CL Spurium:Cyclic CL. Well projected out of surface of the ovary. In buffaloes it is invariably
embaded in nature and creates problem during per rectal examination.

CL Albicans: After regression of CL, a scar like structure is left on the ovary k/a CL albicans. It
is hard scar like and whitish coloured body (White body).

CL Verum: CL of pregnancy. Occupies (75%)area of the ovary, gets embaded in the ovarian
cortex due to its increased weight.

Monotocus species: single ovulation, single offspring e.g. Mare, cattle, buffalo

Polytocus species: many ovulations, many offsprings e.g. Bitch, sow.

Monoestrus species: exhibit one estrous cycle in a year e.g bitch

Polyestrous spp: exhibit continuous regular estrous cycles through the year e.g. Cattle, buffalo

Seasonal Polyestrous spp: exhibit regular estrous cycles in a particular season of the year e.g.
Mare, cat, sheep and goat. Mare is long day breeder whereas goat is short day breeder.

Blood supply:

Utero-ovarian artery-Fallopian tubes and anterior parts of uterine horns

Middle uterine artery: Posterior parts of uterine horns and anterior part of body of uterus
Caudal uterine artery: Posterior part of body of uterus and anterior part of vagina
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Different portions of broad ligament attached:

Mesovarium: Ovary
Mesosalpinx: Fallopian tubes
Mesometrium: Uterus

2. OVIDUCTS (Fallopian tubes/Salpinges):

paired; convoluted tubes extending from near the ovary to the tip of uterine horn. Oviducts
are embedded in the bursa (derived from lateral layer of broad ligament, mesosalpinx).
 Oviduct has three segments; infundibulum(towards ovarian end), ampulla (middle part)and
isthmus(towards uterus). The funnel shaped opening of oviduct towards ovarian end is
known as infundibulum which has fimbria for capturing the ova following ovulation.
 Main functions: picking up of ova following ovulation, gamete transport to site of
fertilization (ampullary isthmic junction), and transport of zygote/embryo towards uterus
for implantation.
 Inflammation: Salpingitis

3. UTERUS
consists of a uterine body and two uterine horns (conua). Types of uterus:
A). Bicornuate uterus(small uterine body and two long uterine horns): cow, sow, bitch,doe and
ewe.
B). Bipartite uterus (prominent body and shorter horns; cruciform or T-shaped): Mare
C). Simple/Simplex uterus: uterus has pear shaped body with no uterine horn. e.g. Human beings
and other primates
D). Duplex uterus: Uterus with two uterine horns each with a separate cervical canal which opens
into vagina e.g Rat, Rabbit, Guinea Pig and other rodents
 Ruminant uterus has mushroom like non-glandular projections known as Caruncles.
Caruncles are arranged in two dorsal and two ventral rows.
 No. of caruncles in different species: Cattle 75-120, Buffalo 60-90 and Sheep 80-90(In
some books 88-96)
Both the horns are joined by dorsal and ventral intercornual ligaments.

Main functions of uterus:

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a)Gamete transport and capacitation: Due to contractions of smooth muscles and uterine mucus
the spermatozoa are transported through uterus to oviduct and the uterine environment helps in
capacitation of sperms
b). Implantation and Gestation: After few days zygote gets attached into uterus and draws
nutrition from uterus. As size of zygote followed by embryo and fetus increases uterus enlarges to
accommodate the developing fetus. Before implantation the embryo takes nutrition from uterine
glands of uterus and the secretion of these glands is known as Uterine milk/Histotroph.
c). Luteolytic functions: Synthesis of PGF2α which is responsible for luteolysis. PGF2α is
derivative of unsaturated hydroxy acids like Arachidonic acid and linolenic acid.
d). Parturition: After completion of gestation myometrial contractions help in expulsion of fetus
e). Serve as site of semen deposition during natural mating in sow and mare.

Inflammation of different layers of uterus:

Endometrium-endometritis, Myometrium(middle layer)-Myometritis, Perimetrium (Outer
layer/serosal layer )-Perimetritis , Uterus (all the three layers)-Metritis and broad ligaments-
Parametritis.

4. CERVIX

A tubular structure with constricted lumen which act as door way to the uterus.
 Cervical canal in cow, doe and ewe has annular rings (interlocking ridges) where as in sow
the canal is funnel shaped and cork screw like
 The annular rings are 3-5 in cattle and 1-3 (mostly only 2) in number in buffalo.
 The cervical canal in mare is more open and having conspicuous folds in mucosa and the
folds projecting into vagina than other farm species.
 Main functions: prevents entry of infection in uterus; transport and store spermatozoa
 In bitch, cervix is poorly defined
 Sow: Cork Screw like penis of boar gets interlocked in cork screw like cervix
 Cervix opens at estrus otherwise closed.
 Cervix projects into vagina like a knob and has a central opening called as external os.
Anteriorily, it opens into body of uterus called as internal os.
 Inflammation:Cervicitis

4. VAGINA

The vagina is tubular in shape and highly musculo-membraneous located dorsal to urinary
bladder in pelvic cavity. The mucosal layer of the vagina is composed of stratified squamous
epithelial cells. These epithelial cells become cornified under the influence of estrogen. External
urethral orifice is the landmark junction of vagina and vulva i.e. vagina is extended from the
cervix to the external urethral orifice.At floor of vagina two ducts, Canals of Gartner (remanant of
wolffian/mesonephric ducts) which open on either side of external urethral opening
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Main functions: it serves as copulatory organ of females, site of semen deposition during natural
mating in cow, ewe and doe and acts as a birth canal and also serves as excretory duct for the
secretions of cervix, endometrium and oviduct.
Inflammation: Vaginitis

5. Vulva
 Also called as external genitalia and consist of vestibule, clitoris and vulvar lips. It acts as
an external opening to reproductive tract. The vestibule is common to both reproductive
and urinary system and contains hymen.Clitoris is located in the ventral commissure and it
is more prominent in mare and sow. Vestibule is homologous to the penile urethra in the
male.The vestibular glands or Bartholian glands(two almond shaped) located under the
mucus membrane in the posterior part of vestibule, are active during estrus and secrete
lubricating mucus.Vestibular glands are homologous to the bulbourethral /Cowper‟s
glands.The luminal space around the external os of cervix is known as Fornix vagina.
Fornix vagina is absent in sow.Hymen(transverse ridge) is located at the junction of
vagina and vestibule which is well defined in the ewe and mare but ill defined in cow,
buffalo and sow. Labia consist of labia minora (homologous to prepuce in male) which is
not prominent in farm animals and labia majora (homologous to scrotum in male).Clitoris
is homologous to glans penis in the male.Upper commissure is acute and separated from
anus by a short distance whereas lower commissure is elongated and bears a tuft of hairs.

ESTROUS CYCLE

The term estrus was used for the first time by Heape (1900). The origin of word estrus is
from a Latin adapted Greek word “Oistros”, which means gad fly/ mad desire. In female cow the
estrus behaviour comprised of attractiveness, proceptivity and receptivity. Estrus is the period
during reproductive cycle when female animals become sexually accessible. Estrous cycle is
defined as time interval between two estrus periods.
Duration of estrus in different species:
Cow/Buffalo: 21 days
Mare: 21 days
Ewe: 17 days
Doe: 21 days
Sow: 21 days
Bitch: 16-56 weeks
Queen: 2-3 weeks (if not mated).
During estrus cow show clear cut behavioural signs especially firm footing and allowing
herd mate to mount on her, the condition is known as “standing heat”. The acceptance of the male
by rage animal during estrum is due to the effect of estradiol on Central Nervous System. Cervix
remain closed except during estrus and secrete cervical mucus which varies with stage of estrous
cycle. It is more at estrus and hangs from vulva as transparent mucus discharge. It has ferning
property (i.e. arborization/crystalization of mucus) if a drop of cervical mucus is examined under
microscope, fern pattern if observed. It occurs due to high chloride present in the mucus under the
influence of elevated estrogen hormone. During other stages it becomes less in amount and thick.
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During pregnancy this mucus forms cervical plug and persists till parturition in order to check
entry of external agents.

STAGES OF ESTROUS CYCLE: 4 stages

1. Proestrus: It starts with regression of previous CL. In this stage there is follicular growth and
no CL. Uterus is slightly tonic, turgid and edematous. Progesterone is low and estrogen is high.
Bleeding occurs only in bitch in this phase.
2. Estrus: it is period of receptivity and animal stands for mating. Duration of estrus in different
species: Cattle/Buffalo: 12-24hrs, Sheep 24-36 hrs, Goat:24-48 hrs; Sow 48-72 hrs; Bitch 9-10
days and Mare 4-7 days. Well developed graffian follicle is present on the ovary. Uterus is
erect, turgid and tonic. Increased secretion of mucus from uterus, cervix and vagina which
hangs from vulva. Cervix is relaxed with open os. Mucosa of vagina is thickened and many
cornified cells being desquamated. During proestrus and estrus the vaginal epithelium is
stratified i.e. multilayered. In other stages and pregnancy the mucosa is 1-2 layered. Similarly
during proestrus and estrus the colour of mucosa is pink to red which due to hyperaemia under
the influence of elevated E2. Vulva is swollen and edematous. In most of the farm animals
ovulation occur in this stage except cattle and buffalo in which ovulation occur 10-12 hrs after
end of estrus (i.e in metestrus). High levels of E2 and low level of P4. In Cattle buffalo, sheep,
goat, mare and bitch ovulation occur spontaneous where as in Cat, Camel and Rabbit ovulation
is coitus induced. Absence of male in induced ovulators may prolong estrus to 7-10 days.
3. Metestrus: is period after estrus for about 3 days wherein CL is formed (except cattle, buffalo).
In some cattle and buffalo bleeding occurs in this phase due to sudden withdrawal of E2 leading
to rupture of capillaries(K/a Metestrual bleeding). Bleeding is not an indication of conception
or conception failure.
4. Diestrus: period when CL is fully functional. This phase starts from day 5 till 17-18th day of
cycle. Fully developed CL is present on 7-8thday of diestrous. Also known as progestational
phase (because of high P4 conc.).
Proestrus+Estrus = Follicular phase and Metestrus+Diestrus =Luteal Phase

HORMONAL REGULATION OF ESTROUS CYCLE

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The estrous cycle is controlled by hypothalamo-pituitory-gonadal axis. Progesterone is the

main hormone regulating the estrous cycle. During luteal phase P4 level is high, due to its negative
feedback on hypothalamus and pituitary it inhibits release of gonadotrophins i.e FSH and LH.
Regression of CL occurs by PGF2α from endometrium.

As CL regress P4 falls and the negative feedback plug is removed which is followed by GnRH
release form hypothalamus. GnRH which acts on anterior pituitary and FSH, LH are released.
FSH act on ovary, cause growth of follicles. When follicle becomes large estrogen is released
from preovulatory follicle, cause preovulatory surge of LH, LH is released from anterior pituitary
and ovulation occurs.CL is formed and if animal is non pregnant, it regresses on day 17-18. The
regressed CL is k/a CL albicans.If animal becomes pregnant CL persists and k/a CLVerum.
Preovulatory FSH, LH control centres in hypothalamus: 1). Preoptic nuclei 2).
Suprachiasmatic nuclei 3)Anterior hypothalamic nuclei
Tonic FSH, LH secretion control centres:1)Ventro-median nucleus 2). Aracuate nucleus 3).
Median eminence

SIGNS OF ESTRUS IN DIFFERENT SPECIES

Cattle and Buffalo: Bellowing, reduced milk yield, reduced appetite, frequent micturition, mount
to other animals or accept to be mounted during standing heat, cervico-vaginal discharge (more
in cattle than buffalo), swollen and congested vulva and increased roaming activity.
Homosexual behaviour is quite frequent in cattle than buffalo. Temporary engorgement of teat
(TET)observed in buffaloes prior to onset of real heat(i.e. in proestrus)is commonly known as
Doka. Doka is used as an important tool for detection of incoming heat by buffalo owners in
non-pregnant animals. Duration of TET is normally 3 days.Sometimes it is also associated with
early pregnancy(Gabh doka).

Mare: Mare is long day breeder and it comes in heat from March to July when days are longer.
Restless and irritable, frequently adopts the micturition posture and voids urine with repeated
exposure of the clitoris (called as “winking of clitoris”). In the presence of stallion mare raises
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the tail to one side and leans her hind quarter and the vulva is edematous with mucoid
discharge.Ovulation takes place on penultimate or last day of heat.

Sheep: Restless, tail wagging and move it laterally, vulva swollen and congested,clear vaginal
mucous discharge and receptive for ram. Ewes seek the ram and together form a following
„harem‟. The ram tries members of this group for receptivity by pawing with a forefoot,
rubbing his head along the ewe‟s side and nipping her wool. Several rams run with a flock, a
hierarchy is established and the dominant male attracts the largest harem.

Goat: The detection of heat in a doe is difficult in the absence of a buck. Edematous and
hyperaemic vulva, tail wagging from side to side and up and down, restless, mounting
behaviour; reduced milk yield and appetite and vocalization (frequent and peculiar type of
bleating).
Pig: Vulva swollen and congested, repetitive grunts, restlessness, by pressing the loin of the sow
with the palms of both hands; the estrus sow will stand motionless (breeding/ mating
stance/back pressure test) with cocked ears, however, an objection will be made by sow those
are not in heat. The pro-estrus female noses the testicles and flank of boar and may mount him
but refuses to be mounted. At the peak of estrus sow assumes a stationary and rigid attitude.
Bitch: Swollen and edematous vulva with sanguinous discharge in pro-estrus, bitch is attractive to
male at proestrus and receptive at the time of estrus.

Important facts:
 Mounting on other animals (early heat).
 Mucus discharge flows as string from vulva to the floor and breaks (early heat).
 Mucus discharge hangs from the vulva to hock and then breaks off (mid heat).
 Mucus string hangs only 25-30 cm long(late heat).
 Within two days of service there is occasional yellowish-white vuval discharge of mucus
containing leucocytes from the uterus.
 Sheep and goat are short day breeder and exhibit estrus during October-February.
 Left uterine horn is longer than the right in camel.
 Quite frequently mare shows evidence of discomfort when the ovary is palpated soon after
ovulation.

MECHANISM OF LUTEOLYSIS
Luteolysis is the degradation of the corpus luteum (just opposite to luteinisation – the
formation of the corpus luteum). Luteolysis occurs at the end of the luteal phase. The process of
luteolysis is initiated under estrogen priming by oxytocin (secreted by the corpus
luteum)and prostaglandin F2a in domestic animals.Progesterone is produced by the corpus
luteum, which inhibits the hypothalamus (the hypothalamus secretes GnRH, therefore
progesterone inhibits GnRH secretion). Initially the oxytocin appears to have no effect, however
after day 16 oxytocin receptors begin to form in cattle. When these oxytocin receptors present in
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endometrium are stimulated by the oxytocin secreted by the corpus luteum, prostaglandin
F2a synthesis and secretion by the endometrium is stimulated.Prostaglandin inhibits the production
of progesterone (which is inhibiting the GnRH secretion and thus preventing the emergence of
new dominant follicles). If progesterone production is inhibited then the oestrous cycle is able to
begin again.Prostaglandin also stimulates further oxytocin release, stimulating more oxytocin
receptors that cause further prostaglandin F2a release. This is known as a positive cascade system
and is used to quickly progress a biological situation, here the situation would be the prevention of
inhibition of progesterone (which is inhibiting GnRH secretion).The reduction of plasma
progesterone concentration means follicular growth can now continue and dominant follicles can
now emerge.
PGF2α synthesized in endometrium which is drained through utero-ovarian vein. Close
approximity of ovarian artery and utero-ovarian vein is important since at these points of
approximation the walls of these two vessels are thinnest, there is no anastomosis. This allows
leakage of PGF2α from utero-ovarian vein into ovarian artery by a counter current mechanism
through the walls of the vessels. It is fairly well evaluated in ewe and cattle than other species.In
mare no local effect and systemic transport of PGF2α takes place.

Counter current mechanism

PATTERNS OF ESTROUS CYCLE

The duration of estrous cycle in buffalo is similar to that in cattle, ranging from 17 to 25
days with a mean around 21 days (i.e. 21±4 days). In majority of cases the normal cycles in cattle
and buffalo lies between 20-24 days. The estrous cycle below 17 days is termed as short cycle and
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may either be due to luteal insufficiency and aberrations in the endocrinological regulation and
secretion of hormones. The estrous cycle length more than 25 days is termed as long cycle. Long
cycles may be of two types:
a) Multiple of normal estrous cycles: It is due to missed heats either due to silent or weak
estruses. This may also because of poor heat detection practices. So double, triple of normal cycles
will be the estrous length e.g. 34, 36, 38, 40, 42, 44, 46, 48 days etc.
b) Irregular Long estrous cycles: The estrous cycle length prolongs, if the death of embryo
takes place after the mid of normal estrous cycle i.e. on day 9-10. If the embryo dies before day 9-
10 of estrous cycle the estrous cycle length is not prolonged.

Important facts:
 Interval between two heats is 7-8 days: Acute endometritis.
 Interval between two heats is 13-17 days: Endometritis.
 In non-pregnant large domestic animals, inflammation of the endometrium due to bacterial
infection can result in significant synthesis and release of PGF2αleading to premature
luteolysis and shortening of the estrous cycle. Thus, short estrous cycles are
pathognomonic signs of uterine infection.
 The luminal space around the external os of cervix is known as Fornix vagina.
 Fornix vagina is absent in sow.
 The reason for „silent‟ first estrus of pubertal animals is because CNS requires to be
primed with progesterone before it will respond and the animal will show behavioural
signs of heat.
 The first ovulatory cycle has been shown to be short in pubertal heifers and the first CL not
only has a shorter than normal life span. But also smaller in size because the dominant
follicle, from which the first ovulation arises, has already entered the static phase of
growth.
 The CL of pregnancy after parturition is progressively invaded by scar tissue and remains
throughout the cow‟s life. On post mortem examination, presence of this corpus albicans
serves to distinguish the cow from heifer and in cow the number of corpus albicans gives
the number of calves born.
 The time required for postpartum uterine involution varies from 4 to 6 weeks (Cattle and
buffalo).
 In large animals after parturition, the ovulation usually occurs from the ovary opposite to
uterine horn which had fetus.

Multiple ovulation and twinning in mare

 Double ovulations occur in up to 30% of estrous cycles depending upon breed and type of
mare(thoroughbreds have higher rate than ponies)
 Twinning is highly undesirable first because very often results in abortion, and second
even if fetus survive is carried to term and many are dysmature, resulting in
dystocia(difficult birth)/neonatal mortality.

Split estrus (False estrus in bitch)

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Most commonly in the first estrus bitches develop vulvar swelling and a serosanguineous
discharge of a short duration. However, ovulation doesn't occur, follicles regress and signs of
proestrus disappear. A normal estrus follows several weeks later. The recognition of split estrus is
important to ensure that mating is achieved at the correct time in relation to ovulation.

Superfetation:is produced when a pregnant female carrying one or more live fetuses comes in
estrus, is bred again a second conception occurs in uterus already containing at least one live fetus.
This is seen in multiparous/polytocus (sow, bitch) and rarely in unipara.

Superfecundation:is produced by a female ovulating two or more ova during one estrus and
copulating with two or more males during that estrus with ova being fertilized by spermatozoa
from each male. It is observed more commonly in multiparous/polytocus especially dogs and cats
because multipara regularly ovulate 2 or more ova, have long heat periods and opportunity for
services by different males are greater than in unipara. Occasionally reported in unipara.

Telogony:It is a misconception that a pure bred animal mated accidently by a mongrel may never
breed true again.

Primiparous: Female animals which are calving first time

Pleuriparous: Female animals which have calved for more than one time.
Nullipara/Nulliparous: Female animal which have never calved.
 Multiparous, polyparous and polytocus are synonyms
 Uniparous, unipara and Monotocus are synonyms
 Uniparous: animals producing one offspring in a gestation
 Polyparous: animals producing more than one offspring in a gestation

REPRODUCTIVE CYCLE IN BITCH

Bitch is a monocyclic animal and there is no frequent, recurring period of estrus. All
bitches have a period of anoestrus or sexual quiescence between successive heats irrespective of
whether they have been pregnant or not. This condition of anoestrous/sexual quiescence is treated
by Prolactin antagonists like cabergoline and bromocreptine. The estrous cycle is traditionally
divided into 4 phases:

1. Pro-estrus: characterised by vulval edema, swelling and a sanguineous/bloody discharge.

The bitch is attractive to males but will not accept the male. Duration of pro-estrus in 9
days.
2. Estrus: Vulva becomes less edematous and vulval discharge becomes clearer, less
sanguinous and less copious. Duration of estrus is 9 days. Allows the male to mount.
Ovulation occurs 1-2 days after the onset of estrus. She remains still while the male
mounts and copulates. During the copulatory tie which lasts for 15-25 minutes, she
becomes restless and irritable. The progesterone levels start to rise before ovulation which
confirms morphological evidence of preovulatory luteinization of mature follicle 60-70
hours before the ovulation. This rise preovulatory P4 rise may provide the stimulus for
bitch to accept the male.
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3. Metestrus: starts when bitch ceases to accept the male; however, there is dispute about its
duration.
4. Diestrus/Anoestrus: At the end of metestrus the bitch passes into a period of anoestrus
without any external signs. The same is also true after parturition following a normal
pregnancy. This phase lasts 2-10 months before the bitch returns into proestrus.

Pro-estrous Estrus Metestrous Diestrus/ Vaginitis/

Cell types Early Late Early Late Anoestrus Vaginitis
Parabasal +++ + - - - ++ ±
Small +++ ++ - - + + ±
Intermediate
Large ± ++ +++ +++ - - ±
Intermediate
Anuclear - ++ ++++ +++ ± - -
keratinised
Red blood + +++ ++ - - ± ±
Neutrophils + - - + +++ + ++++
Changes in cell types and their relative numbers during various stages of estrous cycle in
bitch

 Parabasal cells:Small round cells with round nuclei and small amount of cytoplasm;
Uniform in size and shape.

 Intermediate cells: May be small or large; Round nuclei, nucleus similar in size as
parabasal cells; Entire cell approximately twice the size of parabasal cells; Cytoplasm
becomes angular, irregular and folded as cell enlarges

 Superficial cells: the nuclei become small, pyknotic and fades; Cytoplasm may contain
vacuoles with age; Superficial cells are commonly called cornified cells and Once nucleus
is lost become anuclear cells. Largest epithelial cell as they age and get cornified.
Cornification is the degeneration process.

PSEUDOPREGNANCY/PSEUDOCYESIS/FALSE PREGNANCY/PHANTOM
PREGNANCY/PSEUDOGENETRA/ NERVOUS LACTATION

Pseudopregnancy is very common in bitch, queen and doe and occasionally seen in mare.

Bitch:Pseudopregnancy is believed to be due to intensification and prolongation of metestrus.

There is mammary development and lactogenesis. The bitch will show all the external signs of
pregnancy and will ultimately undergo a mock parturition with nesting, loss of appetite, straining,
emotional attachment to inanimate objects and heavy lactation. Prolactin inhibitors such as
bromocreptine and cabergoline have been used successfully to treat the condition.

Queen (female cat): Sometimes receptors located in queen‟s vulva are stimulated when the queen
is mounted by other females, castrated male cats or by stroking over this area. Sterile matings that
 P a g e | 15

induce ovulation lead to pseudopregnancy. Nesting behaviour and milk production are rarely seen
in pseudopregnant queens but hyperaemia of the nipple will be evident as in pregnancy. The
queen‟s appetite may be increased with some redistribution of fat leading to an increase in
abdominal size.

Doe (female goat): Pseudopregnancy in doe is also known as Hydrometra or cloud burst. It is the
accumulation of sterile secretions within the uterine lumen. Exact aetiology of this condition is not
known but it is always associated with high progesterone levels secreted by a persistent functional
corpus luteum, cessation of cyclic activity and variable degree of abdominal distension. One
possible cause of Hydrometra is early embryonic loss. Two types of pseudopregnancy:

1. Fertilization is followed by early embryonic loss, persistence of CL and doe appears as

pregnant. Abdomen is enlarged and slight udder development. Such type of
pseudopregnancy lasts for the period similar to normal pregnancy or even longer till until
spontaneous regression of CL. At termination of pregnancy large amount of fluid is voided
from uterus and this is known as Cloud burst. Following this abdominal distension
disappears and some does search for the „missing kids.‟
2. When the doe is not mated at estrus, there is cessation of cyclical ovarian activity but no
marked Hydrometra. At the end of the period of acyclicity the affected does expel a bloody
discharge.

MATING AND OVULATION IN CATS

During mounting the tom (male cat) mounts the queen (female cat) and grasps her neck
with his teeth. The queen‟s hind legs paddle as he adjusts his position and this becomes more rapid
during the coitus which lasts up to 10 seconds. The queen cries out during copulation and as the
tom dismounts, she may strike out at him, displaying the typical „rage reaction’. This is followed
by a period of rolling and licking the vulva. As soon as this post-coital reaction has ceased the tom
will usually attempt to mate the queen again and there may be several mating within 30-60
minutes. Receptors are present in the vulva that is stimulated during copulation leading to release
of LH from anterior pituitary. 50% of queens ovulate in single mating and multiple matings may
require to ensure adequate release of LH to induce ovulation. Ovulation is „all or nothing‟
phenomenon.

ESTRUS DETECTION

As per The American Heritage® Stedman's Medical Dictionary, estrus may be defined as
“The periodic state of sexual excitement in the female of most mammals, excluding humans, that
immediately precedes ovulation and during which the female is most receptive to mating.”The
reproductive efficiency depends on, how soon after calving, the cow come into heat and success of
first or subsequent insemination. The ultimate goal of heat detection is to predict actual time of
ovulation.

Methods of estrus detection

1. Record systems: All heats, including those observed in the early post-partum period, must be
recorded and record systems should be used on a daily basis.
 P a g e | 16

(i). Breeding wheel or Herdex record system: These are the wall-mounted reproductive record
systems use colour-coded pins or markings to point out reproductive events for each animal.

(ii). Heat expectancy chart: These are specially designed calendars showing 21 day estrous cycle
so that upcoming heat can be predictable.

(iii). Computer generated action list: Some softwares are available which make a list of animals
those need special consideration or action on a definite period.

(iv). Heat symptoms (Visual Observation): The animals are visually observed for the specific
signs of heat and the signs are different in different species. A best and most common method of
estrus detection may be a direct visual observation of the cow standing firm while being mounted
by other cow.

2. Rectal examination:

Rectal examination is the most efficient method for examination of reproductive tract and by
palpation the internal signs of heat are recorded. During estrus certain cyclic changes occur in the
tract and these may be:

Cervix: soft and relaxed; os of cervix- open.

Uterus: The uterus is erect, tonic and turgid at estrus. Uterine tone can be classified as: i. uterus
flaccid uterus (-), ii. Uterus exhibiting mild response to manipulation (weak tone; +), iii. Curled
and turgid uterus (moderate tone; ++), iv. Uterus very much curled, turgid, ovaries not easily
approachable (intense tone; +++).

Ovaries: Well developed graffian follicle (fluctuating fluid filled structure) is palpable on either of
the ovaries (mostly right ovary).

3. Methods for mount detection (Heat detection aids):

(i). Painted/Chalked Tailheads:This is mainly used by the dairy industry to locate riding cows in
large, confined herds. Tail heads are marked daily by an AI technician. If the chalk is rubbed
off by the next observation, this indicates the cow was ridden and is possibly in heat. Different
colours of chalk are usually used to mark different strings of production. Marking the tail head
with chalk, paint, or crayon and observing for evidence of rubbed off or smeared markings are
less expensive than kamar detectors and have gained popularity in larger herds. This system
works most effectively in loose-housing arrangements where cattle can be restrained in self-
locking head gates to be marked or observed for evidence of smeared or rubbed-off markings,
which indicate the animal was mounted.

(ii). Kamar Heat-Mount Detector: These devices are glued just cranial to the base of the tail.
Pressure from the brisket of a mounting animal requires approximately 3 seconds turning the
detector from white to red from a small storage chamber into a bigger visible plastic
compartment. This timing mechanism helps distinguish between true standing heat versus
false mounting activity. False positive consequences are more common when cattle or buffalo
are restricted in crowded pens or when infested with external parasites rub or scratch their
backs.
 P a g e | 17

(iii) Bovine Beacon: Glued to tail head of cow. It contains fluorescent dye that glows in the dark
when cow is mounted by another cow.

(iv). Novel estrus detection strip: It consists, a reflective material covered by a low-sheen black
acrylic paint. The strip is applied to the sacrum and when the cow is mounted the black
covering is rubbed off. The reflective strip can be observed by using video camera.

(iv). Chin-ball markers on vasectomised bulls: One of the most effective ways of determining heat
is to attach this halter device under the chin of a vasectomised bull. The marker consists of a
paint reservoir with a steel ball valve, similar to a ballpoint pen. When bull rides another
animal, the chin ball marker is activated, and paint marks are left on the in-heat animal‟s back.

Chin ball marker

(v). HeatWatch or ShowHeat: The HeatWatch system for electronically monitoring mounting
activity has been commercially marketed since 1995. This system provides continuous,
radiotelometric monitoring of mounting activity and automated identification of animals.

(vi). Video recording system: This system has been used widely to monitor estrus behaviour,
provided the animal must be identifiable and must be allowed to interact in a loose-housing
system. This system will be successful only if the videotapes are reviewed daily, especially
after the herd has been monitored in the evening. Major disadvantages are the cost of initial
investments for instalment of the device and time to review the recordings.

4. Activity monitors (Pedometer): An important external indicator for incidence of estrus is

restlessness and consequently spend more time to walk and standing instead of resting.
Electronic pedometers are available for automatically recording activity. Activity is measured
by a mercury switch that is turned off and on by movement of the cow. The overall movement
as well as activity of estrus buffalo is increased up to 40% as compared to normal buffalo.

5. Conductivity of vaginal fluids: Conductivity of vaginal mucus changes with the stage of estrous
cycle. Electrical resistance of reproductive tissues and their secretions has been measured and
used as a means of estrus detection and for timing of insemination. The electrical resistance of
reproductive tract secretions is highest during the luteal phase and declines during the follicular
phase of the estrous cycle. The lowest resistance readings occur coincident with the LH surge, a
few hours after the estrogen peak and onset of estrus. After the LH surge, electrical resistance
increases. This tool is labour intensive; washed in disinfectant, and dried prior it is used in
 P a g e | 18

another cow. Without proper cleanliness, the device could spread disease among animals. The
probe is expensive.

Draminski’s heat detector

6. Heat detector animals:

(i).Vasectomised or surgically altered bull: Either a vasectomised bull or bull with a deviated
penis from its original line (surgically altered) could be an efficient heat detector. These animals
are prepared with a chin-ball marker which marks the rump of cows and buffalo those were
mounted during estrus. Bull with surgically deviated penis to prevent intromission may be more
expensive than vasectomised bull, however, this method is favoured since vasectomised bulls can
copulate with cows and there may be chances of disease spread.

(ii). Female with Testosterone treatment/androgenized: Non-lactating culled cows or heifers, even
freemartin heifers can be utilized for the testosterone treatment to make heat detector animals.

7. Laboratory methods:

(i). Progesterone concentrations: Progesterone level decreased during estrus period and its
concentrations remains low and varies between 0.20 – 0.50 ng/ml (always less than 1ng/ml). This
can be detected either by RIA or ELISA method to predict the timing of AI.

(ii). Endometrial biopsy: Phosphates activity increases its peak and persists even 1- 2 hours after
onset of estrus.

(iii). Cervical mucus fern pattern test: Vaginal mucous shows typical fern pattern at the time of
estrus. The fern pattern is seen in Crystoscope (developed by Scientists of IVRI, Izatnagar). It is
available in market with different names e.g. Lykascope (Lyka Health Care Ltd). The fern pattern
can be classified into three types:

 Typical: Clear fern leaf like pattern with well marked branches and has bright and thick
boundaries.
 Atypical: Mixed type appearance. Fern branches remain discontinuous and are not
projected further into branches.
 Nil type: No fern like appearance.
(iv). Vaginal pH fluctuates throughout the estrous cycle but is lowest, 7.32, on the day of estrus in
cattle.

8. Other methods:
 P a g e | 19

(i). Ultrasound scanning: Echo-texture of reproductive organs and presence of mature ovulatory
follicle on any one of the ovary are the characteristic features of estrus cow and buffalo.

(ii). Mechanical nose: Direct electronic sensing system can be used to detect a particular
pheromones secreted by the animals during estrus period. In the near future trained sniffing dogs
may be used for this purpose.

(iv). Estrous synchronization: A definite time of insemination/breeding can be expected by use of

estrous synchronization protocols. This will be discussed in the next chapter.

PUBERTY AND SEXUAL MATURITY

Puberty: It is defined as the age when first estrus is accompanied with spontaneous fertile
ovulation. Puberty in females may be attained when the heifers have gained 35-45% of their
mature body weight but the breeding should be allowed when the female has gained 55% of adult
body weight. The age of puberty is different in different species and breeds. The age is decided
genetically. Before attaining puberty majority of pre-pubertal heifers may exhibit anovulatory or
silent heat. This anovulatory heat is exhibited about 3 months before the actual age of puberty.
Because below this weight if achieves pregnancy later it will lead to dystocia. Puberty should not
be considered as sexual maturity. Sexual maturity is attained at later stage when heifer has gained
mature body weight specific to that particular breed.

Hormonal control of Puberty: Before attaining puberty there is pulsatile release of GnRH from
hypothalamus. It causes release of FSH and LH from anterior pituitary. When puberty is attained
there is increased secretion of GnRH which causes high level of FSH and LH and these results in
induction of estrus. The increased amount of GnRH released at time of puberty is due to decreased
sensitivity of hypothalamus to ovarian steroids. Full reproductive efficiency is not attained in any
species at the first estrus. The age at puberty can be affected by many factors. These factors may
be either genetic or environmental.

a) Breed and genotype: Age at puberty is different in different species because it is decided
genetically. The average weight at the time of puberty depends upon the mature body
weight of that species. e.g. Jersey breed attain puberty at age of 8 months when body
weight is 160kg whereas Holstein Friesen attain puberty at age of 11 months and body
weight is 270kg.
b) Nutrition: If nutrition is not proper the body weight gain is slow and puberty is delayed.
c) Climate: If external temperature is high or adverse then due to low metabolic rate puberty
is delayed.
d) Body condition and sanitation: If hygiene and body condition are not good the animal is
prone to illness and delayed puberty.
e) Vicinity of male: If male is introduced few months before the actual age of attaining
puberty, the female attains puberty at an early stage. This effect is more in sheep, goat and
sow.

Age at Puberty:

Sheep : 7-10 months

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Cattle : 8-12 months

Riverine Buffalo : 15-18 months
Horses : 15-24 months
Doe : 6-8 months
Sow : 5-8 months
Bitch : 6-12 months
Queen : 5-12 months

SEASONAL BREEDING
Regulation in seasonal long day breeders: As the name says these animals specifically
breed during long periods of day light or long photo periods. In the transition of season from the
short periods of day light to long periods of day light, the photo receptors of the eye sensitize the
same and there by the same information is passed to the pineal gland which then removes its
inhibitory action from the hypothalamus. In these long day breeders the inhibitory effect of the
pineal gland occurs during the short days of winter. This is the reason for which mares, stallions
and other equids remain in anestrous during the short days. Once the season changes with
increasing photoperiod into long days, the pineal gland becomes less active and thereby the
secretion of melatonin is also reduced considerably. The inhibitory influence on the hypothalamus
is removed. As a result of which the hypothalamic releasing factors (GnRH) and anterior pituitary
hormones (FSH, LH) are also secreted to initiate the reproductive process for the onset of
breeding. The young ones are produced during spring and summer.
Seasonal short day breeders: Changing photoperiod from longer day light to shorter
days with more periods of darkness initiates the reproductive activity in sheep and goat due to
increase in melatonin secretion. The increased melatonin secretion reaching the hypothalamus,
then stimulates the pulsatile secretion of GnRH. Subsequently, FSH and LH secretion from the
pituitary gland increases, which in turn results in the onset of ovarian activity and the
commencement of the breeding season. Sheep and goat experience an annual period of
reproductive quiescence in response to increased photoperiod. The non-breeding season is
characterized by a reduction in the pulsatile secretion of GnRH, in part because of an increase in
negative feedback activity of estrogen. On the other way changing season from short days to long
days forces them to experience an annual period of reproductive quiescence in response to
increased photoperiod.

FOLLICULAR DYNAMICS

The process of continual growth of ovarian follicle leading to preovulatory size and its
regression or ovulation, is known as follicular dynamics. Follicular development in is
characterized by a wave-like pattern. Immediately after recruitment, a selection phase begins in
which a single follicle emerges from the pool of recruited follicles and continues to grow, whereas
other recruited follicles are inhibited from reaching ovulatory size. This selected follicle grows
faster and is termed as dominant follicle. Next to dominant is sub-dominant follicle. An active
dominant follicle prevents the recruitment and growth of other follicles in the ovary. The
progression of follicular development is a sequence of organized events. The cyclic pattern of
growth of antral follicles is termed as “Wave” and the wave pattern of follicular development has
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been reported in prepubertal, cycling and postpartum animals. Each wave is characterized by wave
emergence, growth, dominance and atresia or ovulation. Once the growth of primordial follicle is
initiated, it is a continuous process until the follicle ovulates or become atretic and finally about
1% of antral follicle ovulates.

Throughout the estrous cycle, during pregnancy and other reproductive stages, there is
continuous follicular activity with growth and atresia. In horse, cattle, sheep, goat and buffalo
follicular development during normal cyclical ovarian activity or in case of camelids during period
of reproductive activity, well organised wave like patterns occurs. In cattle and sheep numbers of
waves are 2 or 3 while in goats it ranges from 2 to 7 with predominantly 4. Buffaloes have
oestrous cycles with 1-wave (3.3 %), 2-waves (63.3%) or 3-waves (33.3%) of follicular growth
with the first wave beginning on day 1, the second around day 9-11 while the third wave appear
on day 17 of the oestrous cycle. There are antral follicles close in size to those which are just
preovulatory, throughout the oestrous cycle including the luteal phase. However, in sow there is
no evidence of a wave like pattern, but the presence of 30-50 intermediate follicles (2-7mm in
diameter), from which on average about 20 which are destined to ovulate start to grow on day 14-
16 of the oestrous cycle, when the CL are starting to regress. Folliculogenesis includes:

1) Recruitment- Gonadotrophin stimulation of a pool of rapidly growing follicles.

2) Selection: A process whereby one or more of the recruited follicles are selected to develop
further.
3) Dominance: The mechanism whereby one (the dominant follicle) or several follicles
undergo rapid development in an environment where the growth and development of other
follicles is suppressed.

Follicular Waves in Cattle

Progesterone
Atresia
Recruitment
Follicular Size

Dominance
Ovulation
Selection

FSH Sensitive Pool

Ovulation 9 16 21
Day After Ovulation
The pattern of follicular dynamics in ruminants can be summarized as follows:
a) Follicles grow in wave like pattern.
b) Periodic surges in circulating FSH are associated with follicular wave emergence.
 P a g e | 22

c) Selection of dominant follicles involves the decline in FSH and acquisition of LH

responsiveness
d) Periodic anovulatory follicular waves continue to emerge until the occurrence of an LH
surge.
e) Progesterone is suppressive to LH secretion and the growth of the dominant follicle.
f) The duration of the inter-wave interval is a function of follicular dominance and is
negatively correlated with circulating FSH.
g) Follicular dominance in all species is more pronounced during the first and last follicular
waves of the oestrous cycle.
h) Pregnancy, the prepubertal period and seasonal anoestrous are characterized by regular,
periodic surges of FSH and emergence of anovulatory follicular waves.

Follicular dynamics in mare

In mare follicular waves have been classified into major and minor waves as follows:

a) Major waves: Follicles diverge into a dominant follicle and sub-dominant follicles as in
case of cattle. These are further sub-divided into primary and secondary waves.
Primary waves: in this the dominant follicle ovulates.
Secondary waves: In this case the dominant follicle is either anovulatory or ovulation is
delayed to after the end of estrus.
b) Minor waves: There is no divergence. Minor and secondary waves tend to occur most
frequently during the transitional phase at the beginning of the breeding season.

Some facts:
 In sow, there is no evidence of wave like pattern, but the presence of 30-50 intermediate
sized follicles (2-7mm in diameter) from which on average about 20 are destined to
ovulate.
 The CL is rapidly formed from the graffian follicle after ovulation primarily from the
granulosa and thecal cells.
 The primary site for initiation of luteolysis is through the large luteal cells which become
smaller, followed by the small luteal cells.
 The onset of heat after foaling occurs on 5-10th day, this first heat after foaling is known as
„Foal heat’. It is traditional to cover (mating in mare is k/a Covering) a mare on day 9 post
foaling.
 Sow exhibits estrus within 48 hours after parturition but there is no ovulation.
 The largest follicle is responsible for most estrogen secretion by the ovary at estrus.
 Stigma formation for ovulation: A thin area of follicular apex, the whole apical wall
become thin prior to ovulation inner layers of the follicular wall protrude through a gap to
form a papilla. Stigma thins out, bulges on ovarian surface, the vascularity of follicular
surface increases except at its centre, which seems devoid of blood vessels and this
avascular area is the future point of rupture. Progesterone is involved in stimulating the
collagenase activity follicular wall. At ovulation the bulging stigma ruptures at the apex,
releasing some of the follicular fluid.PGF2α is involved in follicular rupture and PGE2 in
the remodelling of the follicular layers, terminating in corpus luteum formation. Theca is
predominant site prostaglandin production.
 Endomethacin can block follicular rupture by inhibiting prostaglandin production.
 P a g e | 23

OOGENESIS

It is development and maturation of the ovum. It starts during the prenatal period. During
prenatal period primary germ cells/oogonia are produced from the embryonic gut cells which
migrate to cortex of embryonic gonads. The oogonium is diploid (2N) and formed at day 25-35 of
gestation. Then, oogonia divide by mitosis and produce primary oocytes. The primary oocyte
divide by meiosis but the division is arrested at prophase stage immediately after birth. At the time
of birth in most of the farm animals, the ovaries contain primary oocyte. This primary oocyte from
the birth till the puberty remains dormant and is known as Dictyate oocyte.

When puberty is attained gonadotrophins (FSH, LH) are released which initiate the first
meiotic division (first maturation division) and the primary oocyte result into secondary oocyte
(N) and a polar body. The polar body is retained in the perivitelline space (i.e between zona
pellucida and vitelline membrane). After this division, the number of chromosomes is reduced and
it becomes haploid. Second meiotic division starts immediately after completion of 1st meiotic
division. But it is again arrested at metaphase stage and is not completed till fertilization occurs.
After entry of spermatozoa the second meiotic division is completed and it forms the zygote and
second polar body. So, in farm animals true ovum does not exist because true ovum is a product of
2nd meiotic division which should have occurred before fertilization. In rabbit, ferret, mink and
hamster the ovaries contain oogonium at birth whereas primary oocyte in most of mammals. In
case of bitch and fox, the ovulation occurs at primary oocyte stage whereas in most of mammals it
occurs at secondary oocyte stage. In cow, ewe and sow, the first meiotic division is completed just
before ovulation whereas in mare, it is completed after ovulation.
 P a g e | 24

Oogenesis in farm animals

Time of ovulation and AI/Service:

Species Time of ovulation Time of AI/Service

Cattle and 10-12 hour after end of estrus 10-12 hour after end of
buffalo estrus(mid to end of
estrus)
Mare 1-2 day before the end of 2-4 day after onset of
estrus estrus
Sheep 24-30 hour after onset of estrus 12-24 hour after onset of
(towards the end of estrus) estrus
Goat 24-40 hours after onset of 24-36 hours after onset of
estrus (towards end of estrus) estrus
Sow 30-40 hours after onset of 24-36 hours after onset of
estrus(towards the end of estrus
estrus)

GAMETE TRANSPORT
Transport of ovum and spermatozoa at the site of fertilization is an essential event. The
ovum and spermatozoa are transported in opposite directions. Ovum from the ovary is transported
downward to the oviduct or fallopian tube whereas spermatozoa after ejaculation are transported
upward through the cervix and uterus to the oviduct.
1. Transport of ovum: After ovulation, the oocyte is picked up by the infundibulum which
has ciliated mucosa and the cilia beat toward the direction of uterus. This helps in transport
of ovum towards the ampulla of oviduct. In the ampulla again the mucosa is also ciliated
and the cilia beat towards the uterus. Along with this there is oviductal fluid which also
moves toward the uterus. Due to these movements the ovum passes to ampullary isthmic
junction for fertilization. At this junction ovum remains for 2-3 days.
2. Transport of spermatozoa: The transport of spermatozoa can be:
a). Rapid transport: Due to rapid transport the sperms reach to the internal os within 1.5-3
minutes after mating. This rapid transport occurs due to release of oxytocin which
occurs due to stimulation of external os of cervix either by natural mating or by artificial
insemination. This oxytocin causes contractions of vagina, cervix, and uterus. These
contractions are also caused by PGF2α present in the semen. During rapid transport the
sperms are transported to the oviduct through micelles of cervical mucus. Rapidly
transported sperms don‟t take part in fertilization because they are no capacitated.
b). Colonization of sperm reservoirs: During natural mating billions of spermatozoa are
deposited in vagina in case of cow, buffalo, sheep and goat and cervix acts as the largest
barrier. Massive numbers of sperms are trapped in the complex mucosal folds of the
cervical crypts and cervical mucus also acts as barrier for dead spermatozoa. This
process is facilitated by the fact that the micelles of the cervical mucus direct sperm to
the cervical crypts where the reservoir is formed. The more sperm that enter the cervical
reservoir the more that will reach the oviduct, thus increasing the chance of fertilization.
 P a g e | 25

In artificial insemination cervix is bypassed because semen is directly deposited in body

of uterus. In mare and sow semen is deposited in uterus, therefore the cervix does not
act as a barrier. Besides, cattle, buffalo, sheep and goat, in species in which ejaculation
occurs in the uterus, sperm reservoirs are also formed in the uterotubal junction or in the
endometrial glands in bitch. Almost equal number of spermatozoa are transported to
both the oviducts but slightly more towards the ovulating site. Uterotubal junction is
another barrier after cervix.

Sperm transport through cervix to the uterine lumen

c). Slow release and transport: After adequate sperm reservoirs formation in the
female reproductive tract, the sperms are release sequentially for a prolonged
period. This slow release involves innate motility of sperms and the contractile
activity of the myometrium and mesosalpinx that ensure the continued availability
of sperms for entry to the oviduct for fertilization of the egg.

Egg transport in the oviduct:

In cattle, sheep and swine the transport time ranges from 72 to 90 hours. The unfertilized ova are
retained in the oviduct of the mare for several months.

Fertile life of ovum and sperm (in female genital tract):

Species Sperm Ovum

Cattle 30-48 hrs 20-24 hrs
Horse 72-120 hrs 6-8 hrs
Sheep 30-48 hrs 16-24 hrs
Swine 24-72 hrs 8-10 hrs

Number of ovulations and birth numbers:

 P a g e | 26

Cattle/ Mare Ewe Doe Sow Bitch Cat

Buffalo
Ovulation 01 01 1-2 2-3 10-20 6-8 1-12
rate
birth 01 01 1-2 1-3 6-12 1-12 1-8
numbers

Gestation: It is the period that starts with fertilization or conception and ends with parturition.
Gestation length is longer if fetus is male than female and shorter in case of twins. Early zygote
takes nutrition from the uterine milk/histotroph and after attachment in uterus from the placenta.

Gestation length in different species:

Cattle Buffalo Mare Ewe Doe Sow Bitch Cat Camel

Days 278- 305- 330- 144- 146- 112- 59-68 58-65 375-
293 320 345 151 151 115 415

Bitch: 2 months±2 days

Sow: 4 months±4 days
Sheep/Goat: 5 months±5 days
Cow/Yak: 9 months±9 days
Buffalo: 10 months±10 days
Mare: 11months±11 days
Camel: 13 months±13 days
Elephant: 24 months±24 days

Abortion: The expulsion of dead embryo or fetus that has reached recognizable size is called an
abortion.

In case of cattle and buffalo, it may also be defined as production of one or more calves between
152 and 270 days of gestation; they either are born dead or survive for less than 24
hours.

Still birth: Delivery of dead fetus at full term. It is common in swine and bitch (Polytocus
species).
Hybrids of Mare and …add for external

FERTILIZATION

For fertilization the ovum and spermatozoa should be mature. The ovum is at second
meiotic stage at the time of fertilization and the maturation occurs after fertilization. The
spermatozoa attain maturity in the epididymis and for this the spermatozoa should remain in the
epididymis for 10-15 days to attain the maturity. After ejaculation the female genital tract, the
spermatozoa undergo capacitation. Capacitation is a process by which some components of the
spermatozoa are either removed or modified. The basic purpose of capacitation is to avoid
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premature acrosomal reaction which should occur at the time of fertilization. Acrosomal reaction
is an indicator of completed capacitation. The function of acrosomal reaction is that eggs are
surrounded by glycoprotein coats through which sperm must pass before reaching egg plasma
membrane. Acrosome reacted sperm dissolve coat locally to produce a „hole‟ through which the
sperm swim. Outer acrosomal membrane overlying plasma membrane destroyed (partially or
totally) or become detached from main body of sperm. The acrosomal reaction is a prerequisite for
fusion between ova and spermatozoa plasma membrane and zona free ova cannot undergo fusion
with non-acrosomal activated sperm even through attachment to the membrane surface occurs.

During this acrosomal reaction enzymes like hyaluronidase and acrosin are released from
the acrosome. Fertilization starts with penetration of spermatozoa. First the spermatozoa pass
through the cumulus cells and the penetration is facilitated by the hyaluronidase enzyme in case of
cattle and arylsulfatase in sow. After this, the spermatozoa reach the zona pellucida. At the zona
pellucida, it attaches with receptors. The receptors are species specific i.e the spermatozoa of same
species attaches with the ovum of same species. Three glycoproteins, ZP1, ZP2 and ZP3 are
synthesized by maturing oocytes. These are present in all mammals but with variation in these
proteins. ZP3 functions as sperm receptor to which only sperm with an intact acrosome can bind.
Binding of sperm to sperm receptors occurs through an interaction with O-linked oligosaccharide
on ZP3. Then, the spermatozoa pass through the zona pellucida and get attached with vitelline
membrane and the penetration is helped by acrosin enzyme. Once it attaches with the vitelline
membrane zona reaction occurs. During this reaction certain components from the zona membrane
are released and this causes reorganization of zona membrane and it prevents entry of other
spermatozoas i.e cortical reaction or polyspermy block.

Cortical reaction or polyspermy block: Block to polyspermy is at zona pellucida in most

mammals (e.g. sheep and swine) with a secondary physiologic block at vitelline membrane in
rabbit (Vitelline block). Initial polyspermy block is at sperm penetration of ovum when cortical
granules are released into perivitelline space. “Release of contents of cortical granules cause
extensive reorganization of zona pellucida and/ or vitelline surface which cause hardening of zona
pellucida and inactivation of ZP3 receptors to prevent polyspermy, is termed as “cortical
reaction/Polyspermy block”

The spermatozoa through phagocytosis digest the vitelline membrane and enter the
cytoplasm of the ovum. Immediately after this vitelline block occurs. Again, in this certain
substances are released by the cortical granules of vitelline membrane and it prevents fertilization
by other spermatozoa.
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In this process, the tail of sperm is separated and get degenerated. Subsequently, the
cytoplasm of the ovum shrinks and it forms female pronucleus. The head of spermatozoa forms
male pronucleus. Both the pronuclei migrate towards the centre of ovum, come in contact with
each other; their membrane are dissolved and the mixing of their contents takes place. The fusion
of male and female pronuclei is termed as Syngamy. After, this fusion the second polar body is
released and fertilization process is completed.

CLEAVAGE

During this stage the zygote divide by several mitotic divisions (always). The zygote or
one celled stage is quite large having a low nuclear to cytoplasm ratio. To attain a ratio similar to
somatic cells, cell divisions occur without an increase in cell mass. The process is referred to as
cleavage. By first mitotic division a two celled embryo is formed and each cell is known as
blastomere. Then, due to subsequent divisions a 4, 8, 16, 32 and 64 celled embryo is formed. The
8-16 celled stage embryo is known as Morula. Later, in the morula a fluid filled cavity called
blastocoel is formed and this embryonic stage is known as Blastocyst. At this time blastomeres
flatten on each other to form a round embryo and internal cellular components, and surface
microvilli become asymmetrically positioned in a process termed polarization. The combined
processes of flattening of the blastomeres and polarization are referred to as “compaction‟. In
sow, blastocyst is formed on day 5, in mare and sheep on day 6 and in cattle on day 7. In cattle 16-
32 celled embryo enters the uterus on day 3 or 4, days 5-8 in bitch and queen, whereas in sow a 4-
8 celled stage embryo enters the uterus on day 2. In ewe (8 celled stage) it happens on day 3. The
tubal transport of fertilized eggs of mare probably takes 5-6 days, by which time they are at the
blastocyst stage. The egg is transported to the uterus from the oviduct to the uterus irrespective of
fertilization except in mare wherein the unfertilised egg remains in oviduct for several months. In
the uterus the early zygote gets its nutrition from the uterine secretion known as
Histotroph/Uterine milk. The blastocyst is contained in the zona pellucida and due to further
divisions it elongates due to which zona pellucida ruptures and the blastocyst comes out. This is
known as zona hatching and it occurs in uterus 4 to 8 days post ovulation. Afterwards, the
elongation is very fast and blastocyst starts differentiating into extra embryonic-membrane from
which the placenta develops. Also, there is formation of germ cell layer in the blastocyst. The
germ cell layers are ectoderm, mesoderm and the endoderm. From the ectoderm the central
nervous system and external organs like hair, eye, ear, nail and sweat glands develop. From the
mesoderm all the internal organs except digestive system develop. From the endoderm, the
digestive system, liver, lung and pancreas develop. The organogenesis occurs during 2 nd to 6th
weeks and on day 21 the heart beat starts in the embryo.
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The development of fetus in fetus can be divided in to three periods:

1. Ovum period: The period from fertilization till implantation or attachment.

2. Embryonic period: It is the period from implantation till differentiation or organogenesis.
In Cattle, it is the period from 15-45 days
In Sheep, it is the period from 11-34 days
In Mare, it is the period from 12-55 to 60 days
3. Fetal period: It is the period from differentiation till parturition.
In Cattle, it is the period from day 45 till parturition
In Sheep, it is the period from day 34 till parturition
In Mare, it is the period from day 55 till parturition

 Prostaglandins of F series, acting locally appear to inhibit transport of embryo into the
uterus whereas prostaglandins of the E series appear to accelerate their delivery to the
uterus.

Polyspermy

It occurs due to fertilization by more than one spermatozoas. It is due to ageing of the
gametes. Due to this polyploidy occur. The zygote may survive for some time but early embryonic
death occurs. Physiologic polyspermy is common in birds and reptiles; the incidence of
polyspermy in most mammalian species is only 1 to 2%. The Pig appears most susceptible to
polyspermy, especially as a result of delayed mating or insemination up to 15% of the eggs are
penetrated by more than one spermatozoa.

Ageing of gametes

It occurs if the animal is not inseminated at proper time. If insemination is too early then
spermatozoa become older or aged. If inseminated too late then ageing of ovum occur. Due to
ageing polyspermy may occur.

Parthenogenesis

It is the development of egg without fertilization. It is common in invertebrates. If the egg

is subjected to electric stimulation or hyaluronidase treatment it may develop up to blastocyst
stage. In mammals due to ageing the ovum may develop up to second polar body stage.
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Twinning

It is natural and common in sheep and goat. In mare about 30% of females double
ovulations take place. It twinning is there, then in majority of cases either one fetus dies or both
get aborted. Rarely both are born alive. It can be diagnosed by per-rectal examination or
ultrasound and the one of the embryonic vesicle/fetus is destroyed manually. In cattle it is rare and
can occur in 0.5-2% of cases. It is highly undesirable in monoovulatory animals and may lead to
dystocia and retention of placenta. Twins can be:

a) Dizygotic: These develop from two separate fertilised eggs ovulated during the same
estrous cycle. These are relatively common and may be of same or opposite sex.
b) Monozygotic: These develop from single fertilised egg. These are always of same sex and
are phenotypically and genetically similar.

Twinning may occur due to separation of blastomeres and up to 16 cells stage is

blastomere is totipotent means capable of developing into an independent living being or it
may develop due to formation of two inner cell masses in the zygote. As compared to
dizygotic, monozygotic are less common because the chances of abortion are more in
monozygotic. With twin pregnancy in cattle, fusion of chorioallantois of adjacent conceptus
results in a common blood circulation. Therefore, around 95% of heifers born co-twin with a
male are sterile freemartins.

Freemartinism is a distinct form of intersexuality that arises as a result of vascular

anastomosis of the adjacent chorioallantoic sacs of heterozygous fetus. The vascular
anastomosis occurs as early as 30 days of gestation. If there is death of male twin of a
heterozygous pair after this time with the other being carried to term. Therefore, it is possible
for a single born freemartin to occur. The external genitalia of freemartin heifer may appear
normal; the internal genitalia are grossly abnormal. The gonads are typically vestigial but in
minority of cases have undergone masculinization and the gonads resemble testes
(parenchyma contains recognizable tubules and interstitial tissue). Structures developed from
paramesonephric duct are absent or hypoplastic. Development of mesonephric ducts is related
to the degree of masculinization of gonads. Vestigial gonads of freemartins are devoid of
follicle and oocytes.

The newly born freemartin can be recognized by prominent clitoris with an obvious
tuft of hairs at the ventral commissure of vulva, although these signs are not always reliable. In
adult female the length of vagina is about 30 cm compared with 8-10 cm in freemartin. In
calves of 1-4 weeks of age the vagina is normally 13-15 cm in length compared with 5-6 cm in
 P a g e | 31

a free martin. The most accurate method of diagnosis is demonstration of sex chromosome
chimerism in cultured lymphocytes.

Intrauterine migration of embryos

In polytocus animals (sow & bitch) and in mare, it is common. It is essential for survival of
fetus. After entry into the uterus the free zygote move from one uterine horn to the second and
there is mixing of zygotes of both the uterine horns (in sow & bitch). By day 12, the zygotes
become stabilized and are equally distributed between both the uterine horns. In mare, the zygote
migrate about 10-14 times daily between 12-14 days. By day 25 it become stabilized and usually
found in the uterine horn which is opposite to the corpus luteum. In cattle, the migration does not
occur. But, in sheep if there are twins then zygote migrate from one to another horn and in case of
single zygote no intrauterine migration occur.

Implantation

It is the attachment of embryo with the endometrium of uterus. The trophoblast of embryo
attaches with uterus by secreting proteolytic enzymes. In large animals the attachment is
superficial and non-invasive. In sow, attachment starts on day 12 completed by 18 and attachment
is by diffused microvilli.

Maternal Recognition of pregnancy

After implantation certain signals are produced by the fetus which prevents regression of
CL. So, the critical period during which the signals are passed to the dam or female is known as
Maternal Recognition of Pregnancy (MRP). In different species different substances are produced
by the developing embryo which helps in maintenance of pregnancy. Initially, the conceptus of
domestic farm species must physically cover a large portion of maternal endometrium to regulate
release of PGF2α to prevent luteolysis.

Species MRP Day of definite attachment

Sow 12 18
Ewe 12-13 16
Cow 16-17 18-22
Mare 14-16 36-38
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In sheep, between days 12-21 a protein called Interferon- τ/OTP-τ (Ovine Trophoblastic
Protein Tau, Mol weight 18-20 kDa) is produced which again prevent the synthesis of PGF2α.
OTP-tau secreted by embryo prevents the rise in endometrial oestrogen-α receptors that precedes
the rise in endometrial oxytocin receptors. The consequence is that OTP-τ indirectly suppresses
the expression of endometrial receptors. Oxytocin of luteal and possibly of central origin can thus
no longer bind to the uterus, and this result in a reduction in the pulsatile secretion of PGF2α.

In sow, by the day 12, the embryos are more or less becomes evenly distributed and this
process is known intra-uterine spacing. The estrogen production by conceptus plays a vital role in
the maternal recognition of pregnancy and the extension of the life span of CL. The administration
of exogenous estrogens parentrally in non-pregnant sows prolongs the life span of the CLs (i.e
luteotrophic) and extends the interoestrous interval. After the initial secretion of estrogen at day
11, a second sustained release of estrogen by the conceptus is necessary between day 14 and 18
for luteal persistence beyond day 25. These estrogens do not prevent the synthesis of PGF2α but
cause its sequestration into uterine lumen and thus prevent its entry in uterine vasculature
(inhibition of endocrine action). Sow conceptus also produces interferons (Type I and II) at the
time of elongation. Other important factor for maintenance of pregnancy in sow is the number of
foetuses present in the uterus. To maintain pregnancy in sow, there should be atleast two foetuses
in each uterine horn.

In mare, between days 10-14 the embryo migrates 12-14 times daily which prevents the
synthesis of PGF2α.

In cows, between days 16-17 bovine Trophoblastic Protein (bTP-1, Mol. weight 24kDa)
produced by the embryo which prevents the synthesis of PGF2α from the endometrium of uterus.
bTP-1 shows immunological cross-reactivity with oTP-1 as well as having a high amino acid
sequence homology with both oTP-1 and interferon-α (Interferon- α), it also possess antiviral
activity. Bovine Trophoblastic Protein is also known as bovine interferon- τ (bIFN- τ). This exerts
its antiluteolytic effect by modifying oxytocin receptors, thereby inhibiting the synthesis from
Arachidonic acid and subsequent release of PGF2α.

Placental attachment in ruminants is characterised by appearance of binucleated cells

arising from uninucleate cells of the trophoblast. Binucleated cells appear on the day 17 and are
present throughout the gestation. These cells migrate and fuse with the underlying uterine surface
epithelium to form multinucleated cells or a synctium. These synctium may be involved in
immunologic protection of the conceptus and the transfer of the placental lactogen synthesized by
the binucleated cells into the maternal vascular circulation.
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Placenta

After attachment of fetus within uterus, it gets nutrition from the dam. For this specialized
parts of fetus come in contact with uterus. The placenta is formed by the union of trophoblast with
the endometrium. The main functions of the placenta are:
a). It protect the fetus from the external physical injury.
b). It stores the fetal waste products in the allantoic fluid.
c). It synthesise many enzymes and hormones which helps in maintenance of pregnancy.
d). It provide nutrition to the fetus from the dam.

In general placenta can be:

1. Deciduate: The attachment of trophoblast within the endometrium is deep and firm. At the
time of expulsion of placenta, a part of endometrium is lost and there is bleeding. It is seen
in bitch, cat, rodents and primates.
2. Non- Deciduate: It is observed in most f the domestic animals. The attachment of
trophoblast is superficial, so part of endometrium is lost at the time of expulsion of
placenta and no bleeding takes place.

On the basis of distribution of chorionic villi the placenta can be:

1) Diffused: It is found in mare, sow and camel. Chorionic villi are present throughout the
chorion and all the villi get attached with endometrium. The villi are absent in the area
just opposite to the cervix. This area is known as cervical star. In sow, the villi are
present in a radiating manner and the circulating areas containing villi are known as
Areolae. The areole are present opposite to uterine glands for absorption of nutrients.
Uterine glands are devoid of any attachment.
2) Cotyledonary: It is also known as Placenta multiplex. It is seen in cattle, buffalo,
sheep and goat. Initially, villi are present throughout the surface of chorion and later
villi are restricted to opposite to caruncles and rest of the villi get degenerated. These
villi form cotyledons. These villi form cotyledons which get attached with caruncles
and the union results into placentomes. Placentomes are convex in cattle and concave
in sheep.
3) Zonary: It is found in bitch and cat. The villi are present in zones in a diameter of 1-3
inches.
4) Discoidal: It is found in human beings and monkey. The villi are present in an oval
disc like structure.
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Histologically, the placenta can be:

1). Haemochorial: It is observed in human being and monkeys. The chorion of fetus
directly contact with blood of uterus. Blood is lost at the time of separation of placenta in
these species.

2). Endotheliochorial: It is observed in bitch and cat. The chorion of fetus is attached with
endothelium of blood vessels of uterus.

3). Epitheliochorial: It is found in mare, sow, and camel. Chorion of the foetus is attached
with the endometrium of uterus.

4). Synepitheliochorial/Syndesmochorial: It is found in cattle, buffalo, sheep and goat i.e

in ruminants. Placental attachment in ruminants is characterised by appearance of
binucleate cells arising from uninucleate cells of the trophoblast.

DIFFERENT PROTOCOLS FOR INDUCTION AND SYNCHRONIZATION OF ESTRUS

Estrus synchronization

Manipulation of estrous cycle or induction of estrus to bring a group of females into estrus
at predetermined time is termed as synchronization of estrus. Ideally an estrus synchronization
system should elicit a fertile, tightly synchronized estrus response in a high percentage of treated
female. The pharmacological control of estrus cycle is based on two approaches:
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1. Either shortening the life span of CL by inducing early luteolysis by using PGF2α
Or
2. Extending the life of CL by administering the Progesterone

1. By shortening of luteal phase: This induces premature regression of a cyclic corpus luteum
(CL) known as luteolysis. Two primary luteolytic agents that are prostaglandin F2α or its
analogues and Estrogen administration result in estrus and ovulation within 2-3 days. In all
species, the CL is quick to respond for luteolytic agents during definite stages of its
development. Luteolytic agents will not cause regression of the CL during the first 12 or 13
days of the cycle in the pig and first 6 days of the cycle in horses and ruminants. Estrus and
ovulation can also be synchronized in animals through a combination of progesterone and a
luteolytic agent. Basis of use of these drugs are: luteolytic agent to regress the CL and the
progesterone prevent estrus until its withdrawal.

2. By extending, luteal phase: This method involves prolong administration of a progesterone

therefore; CL regresses naturally during the period of administration of progesterone. This
approach, results in a negative feedback on LH secretion after regression of the CL. Following
withdrawal of progesterone, follicular growth, estrus, and ovulation occur within 2 to 8 days.
The interval from removal of progesterone to the onset of estrus varies among species and the
methods of progesterone treatment within species. Generally, long-term progesterone treatment
is to be given for 10 days or longer depending on the species.
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Synchromate B: Implant contains 6 mg of norgestomet

Injection contains 5 mg estradiol valerate and 3 mg norgestomet
Injection is administered at the time of implant insertion.
Crestar: Implant contains 3 mg of norgestomet
Injection contains 5 mg estradiol valerate and 3 mg norgestomet
Injection is administered at the time of implant insertion.

SUPEROVULATION AND EMBRYO TRANSFER TECHNOLOGY/MULTIPLE

OVULATION AND EMBRYO TRANSFER (MOET)

Assisted Reproductive Technology (ART): All treatments or procedures that include the in vitro
handling of human/animal oocytes and sperm or embryos for the purpose of establishing a
pregnancy.

Embryo Transfer Technology (ETT) is a technique used to increase the reproduction rate of
cows and buffaloes. Embryo transfer simply means collection of an embryo from a superior donor
female and its transfer to the uterus of a recipient female of the same species

Superovulation

This is the process of induction of multiple ovulations, above the normal level that occur
naturally, using exogenous hormones and it can be determined by the status of follicular
 P a g e | 40

development at the time of gonadotrophins treatment. Three or more than three ovulations (≥ 3CL)
are considered as superovulatory response. In cattle and buffalo, superovulation protocol during
mid-luteal phase (9 to 12 days) has generally given better results. Mainly FSH and PMSG are used
to perform superovulation. As FSH has a biological half life of only 2-4 hours, repeated injections
are required at 12 hourly intervals to maintain the required blood levels, however, PMSG has
about 7 days biological half life. Therefore, single dose of PMSG is administered. In comparison
to PMSG, FSH is costly but most FSH preparations have a more consistent FSH: LH ratio.

PMSG has both advantages and disadvantages for the use.

Advantages:

(i). It has longer biological half life so there is no need to administer at regular interval. (ii). This
drug is relatively less expensive than FSH.

Disadvantages of PMSG

Since, it exhibit activity as both FSH and LH, therefore, it has variable and unpredictable FSH:
LH ratio and residual amount of PMSG may have a continuous stimulatory effect post-ovulation
and consequently cause development of post-ovulatory estrogen producing follicles. Extended and
prominent estrogen production may interrupt conception and early embryonic development.

Limitations of superovulation

(1).The greatest problem with superovulation is the large degree of variation in superovulatory
response between individual of the same species.
(2). No reliable way of predicting the number of oocytes in response to the gonadotropins.
(3). Difference in potency, purity and quality of commercially available preparations may affect
the response to superovulation.
(4). Breed differences and seasonal effect also contribute to variability and older cow appear to be
less responsive for gonadotrophins.
(5). Repeated treatment with gonadotropins may induce the formation of antibodies to the
gonadotropins, resulting in lower number of oocytes ovulated with each successive treatment.
(6). Another possible consequence of the immunogenicity of gonadotropins is anaphylaxis upon
repeated treatment.

Criteria for selection of donor:

1) Superior individual performance.
2) Good reproductive performance.
3) Regular cyclicity.
4) Ovaries must be free and without adhesions.
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5) Cervix must be easy and allow free passage of the catheter without any kink and
abnormality.
6) Fallopian tubes and uterus also must not have any abnormalities and infections.
7) No detectable genetic defects in the donor.
8) Comparatively younger donors are preferred.
9) Donor must have calved atleast 60 days back.
10) Best time to initiate superovulatory treatment is 90-100 days post calving.
11) For repeated superovulation donors should be treated atleast 60 days after 1st
superovulatory treatment.

Some superovulatory regimes in cattle and buffalo in donors along with synchronization in
recipients

25 mg Lutalyse i.m in 2 & 3

protocol on day 13 (morning)

* Schedule for each treatment protocols.

 Dose of Dianaprost (Natural prostaglandin; LutalyseR ) is 25 mg (5ml)

 Dose of Cloprostenol (Synthetic prostaglandin; PragmaR, VetmateR) is 500µg (2ml)
 The day of estrus before treatment is treated as day „0‟. And after treatment, the day of
estrus is treated as day‟0‟. AI is done on day 1 post estrus and embryo flushing on day 7.

Remarks on FSH protocol:

 Constant or tapering dose schedule of FSH can be used.
 Treatment may be started in between day 10-12 of the cycle.
 Three or four days treatment may be done.
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 PGF2α may be given on 3rd or 4th day morning i.e. 48 or 72 hours after initiation of FSH
treatment.
 Embryo recovery and their quality are usually poor than with the use of FSH-P in cattle
 In buffalo, ovulation rate, superovulation response, embryo recovery and their quality have
been found low using PMSG than FSH-P. Overall response of superovulation and recovery
of transferable embryo per donor have been low in buffalo than cattle.

SUPEROVULATORY ESTRUS

Following administration of prostaglandins (PGF2α) i.m, donors are subjected to detection

of estrus twice during morning and evening using a teaser and visual observation of signs
of estrus. Majority of donor (80-90%) exhibit estrus 36-48 hours following administration
of PGF2α. Intensity of estrus and estrus response has been found greater than non-
superovulated control animals. Mean interval from PGF2α injection to onset of estrus is
shorter in superovulated donors than normal recipient females. Recipients are given PGF2α
12-24 hours earlier than donor for getting efficient synchronization of estrus between
donor and recipients for embryo transfer work.

BREEDING OF DONOR

Confirm estrus on per-rectal examination by observing opening of external os and

relaxation of cervix, turgidity and estrus tone of uterus. Donors are inseminated twice at
10-12 hours interval. High quality semen with double dose should be used for AI (twice
dose of semen than normal for fertilizing more number of oocytes released by the rupture
of multiple follicles induced due to gonadotrophin treatment). Semen should be deposited
in both the horns. Therefore, only skilled and trained personnels should be allowed to
inseminate the valuable donor.

SUPEROVULATION PROTOCOL FOR SHEEP AND GOAT

 Selection of donor
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 The day of estrus is taken as day „0‟. Total dose of FSH is 20IU. Prepare six injections of
4/4, 3/3 and 3/3 IU in separate syringes and keep in deep freeze. Start the treatment on day
10.
Day Morning Evening
Day 10 4IU 4IU
Day 11 3IU 3IU
Day 12 3IU+50 µg Cloprostenol, 3IU
Day 13 Observe the signs of estrus
Day 14 Donor will be mated in standing. This is treated as day „0‟.
(day 0)
Day 1 Mating , Inject 500 IU hCG or 50µg GnRH at mating
Day 3 Surgical collection of embryo from fallopian tube. Expected stages of
embryo are 4,8 and 16 cell
Or Surgical collection of embryo from the uterus. Expected stages of
Day 5 embryo is morula

METHODS OF EMBRYO COLLECTION AND TRANSFER

Embryo can be collected in cattle and buffalo by surgical and non-surgical methods.
Surgical method is more complex, full of post-operative complications and invariably leads to
ovario-bursal and uterine adhesions. For these reasons, it is not practised for routine collection of
embryo in cattle and buffalo. The non-surgical method of embryo recovery is preferred in cattle
and buffaloes.

(1). Non-surgical methods: (2). Surgical method

(1). Non-surgical methods: (A). Trans-cervical method (B). Laparoscopic method

(A). Trans-cervical method: This method is widely used for large animals e.g. Cattle, Buffalo
and Mare by using 3-way Foley‟s catheter.

i. After epidural anaesthesia, Foley‟s catheter is placed through cervix, guided by stilette.
Catheter may be placed either in the uterine body or in one horn.

ii. Following positioning of catheter in one horn, the balloon is inflated by pushing air about 30-
50cc with the syringe, so that catheter may be fixed at position.
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iii. Flush the uterine horn by injecting luke warm phosphate buffer saline (PBS) 30 to 60ml.

iv. Massage the uterus through rectum and allow the infused PBS to flow in a collection vessel.

v. Repeat the procedure until 300 to 800 ml of medium has been used.

vi. Follow the same procedure for other uterine horn.

Note: Same procedure is involved in case of mare, except the balloon is to be inflated in the cervix
and both horns are flushed simultaneously.

Fig.: Trans-cervical method of embryo collection

(B). Laparoscopic method: McKelvely et al., (1986) first reported the use of laparoscopy under
general anaesthesia for collection of embryos from the uterus of sheep. Subsequently, this method
has been used in goat and pig. Instrument for flushing are to be inserted through stab incision in
comparison to mid ventral incision to be made for surgical method.

i. Insert the laparoscope through one stab incision in the skin.

ii. While visualizing the uterus, insert 2-way Foley‟s catheter into one uterine horn through
another stab incision.

iii. Insert an i/v catheter into the uterine lumen close to the uterotubal junction.
 P a g e | 45

iv. Flush the uterine horn by injecting about 40 to 50ml of flushing medium and collect it in a
sterile vessel.

v. Repeat the same procedure for other uterine horn.

(2). Surgical method:

This method is mostly used for embryo collection in small animals e.g. sheep, goat and pig
by exposing the uterus through mid-ventral incision under general/local anaesthesia. Animals are
fasted for 24 hours before operation and the area cranial to the udder is shaved. Uterine horns are
exposed through 8-12 cm long incision between udder and umbilicus. The flushing medium, PBS
is infused to into the base of the uterine horn and flushed toward the uterotubal junction.
Afterwards the flushing medium is collected through a small glass tube or a blunt syringe needle
inserted into the uterine lumen. This procedure may also be carried out in the reverse way. A
volume of 2 to 20ml is used to flush the fallopian tube, while 10ml is used to flush the uterus
depending on its size.

After collection of embryos, it is sieved with the help of sieve of having pore size of 70
microns. The contents of the filters are poured in to Petri dishes (100 X 15mm size) marked with
grids on the bottom. The filter sieve is cleaned with PBS and contents are poured into the
searching dish. The isolation of flushed medium is done under low power (10-15X) of zoostereo-
microscope. An average of 7-10 embryos are collected from each flush. Embryos are microscopic
in size (about 0.2 mm) and only undamaged embryos at proper maturity should be transferred.

Under most conditions, embryos between the eight cell (four cells in pigs) and
blastocyst stage result in the highest pregnancy rates. Older embryos may tolerate in vitro
handling better than younger embryos. Most bovine embryos are collected from the uterus from
the morula to the expanded blastocyst stage. In general, only excellent and good embryos are
considered as transferable embryos. Defective embryos showing following morphologic
anomalies must be discarded:
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i. Blastocyst of variable, non-uniform size

ii. Cellular debris in the morula.
iii. Degenerated blastocyst within oblong-shaped zona pellucida.
iv. Disintegrating mitotic figure.
v. Indistinct foamy blastomeres.
vi. Fragmentation of cytoplasmic and nuclear material.
vii. Abnormal shape of morula or blastocyst.

PROCEDURE INVOLVED FOR EMBRYO TRANSFER

Cattle and Buffalo:

In these species mostly trans-cervical approach is used. Epidural analgesia is used to

control the animal. Then, recipients are per rectally examined for determining the presence of
corpus luteum on the ovary. Straw containing embryo is fitted into the Cassou AI gun and covered
by sterile sheath under aseptic condition. Then gun is inserted into the uterine horn ipsilateral to
the corpus luteum and deposit the embryo.

Sheep and Goat:

Embryo transfer is conducted under local or general anaesthesia through mid ventral
laparotomy. For embryo transfer, the tip of a capillary pipette containing the embryos is inserted
 P a g e | 47

in the oviduct through the infundibulum to deposit embryos into the ampulla. When embryo is to
be transferred to the uterus, the uterine horn is punctured with a blunt needle, and the embryos are
expelled from the tip of the capillary pipette inserted into the uterine lumen. Laparoscopy may
also be used to transfer of embryos into the uterus.

MILESTONES IN EMBRYO TRANSFER TECHNOLOGY IN ANIMALS

Year Species Event Researcher

1890 Rabbit Birth of an offspring from embryo transfer Heape
1949 Sheep and Birth of a lamb and a kid from embryo transfer Warwick and
goat Berry
1951 Cattle Birth of calf from embryo transfer Willett et al
1973 Cattle Birth of calf produced from frozen embryos Wilmut and
Rawson
1982 Cattle Birth of calf from IVF Brackett et al
1983 Asian Birth of buffalo calf from embryo transfer Drost et al
buffalo
1996 Sheep Birth of lamb cloned (Dolly) from adult somatic cells Wilmut et al
 P a g e | 48

India
1988 Buffalo Production of the first buffalo calf following ET in Misra et al.
Asia/India
1988 Cattle Production of first cow calf following embryo Totey et al.
transfer in India at NII

Disadvantages of ETT:

1. Increased expenses and higher breakeven costs for calves.

2. Requires a higher level of management.
3. Increased potential for spread of certain diseases.
4. Not all potential donors respond positively to treatment.

PREGNANCY AND ITS DIFFERENTIAL DIAGNOSIS

The pregnancy diagnosis is also known as Cyesiognosis and is necessary for cost-effective
animal husbandry. In the current systems of planned breeding, diagnosis of pregnancy would help
to evaluate the therapies at an early date and devise alternative manipulations. In some situations,
in the pet practice pregnancy may not be desirable by the owners and an early diagnosis would
help in termination of these unwanted pregnancies. An early pregnancy diagnosis is essential in
mares to tease them if non pregnant, and try to get them pregnant in the same season. It therefore,
appears that early diagnosis of pregnancy is essential in animal management for economic
reasons. For an economical dairy farm, cows must calve every year, and to maintain this sequence,
identifying pregnant animals at an early date seems imperative. Methods of pregnancy diagnosis
in the domestic animals can be classified into visual/managemental, clinical and laboratory
methods. In most of the domestic animals clinical methods are currently used.

I. Visual/ Managemental methods:

A). Non return to estrus
B). Udder development

II. Clinical methods:

A). Trans-rectal palpation
B). Ultrasonography
C). Radiography
D). Abdominal ballottement /Palpation
E). Laparoscopy

III.Laboratory methods:
A).Vaginal biopsy
B). Immunological methods
i. Progesterone hormone assay
 P a g e | 49

ii. Estrone sulphate

iii. PMSG
iv. Pregnancy associated glycoproteins (PAG)
C). Biological methods

I. Visual/ Managemental methods:

A). Non return to estrus:

When an animal is mated and it does not return to estrus the owner usually thinks that the
animal has become pregnant and hence has not returned to estrus. This happens because during
pregnancy, the conceptus inhibits the regression of the corpus luteum and thus, prevents the
animal from returning to estrus. However, many a times the animal does not return to estrus
because of non regression of CL due to reasons other than pregnancy. Moreover, in the seasonally
breeding species the animal may not return to estrus (when mating is done during the end of the
breeding season) because the season was over. Anestrus and the rare occurrence of gestational
estrus in cattle and buffaloes can affect the reliability of non return to estrus as a method of
pregnancy diagnosis. Moreover, difficulty in estrus detection and silent estrus render this method
of pregnancy diagnosis unsuitable for the buffalo. Therefore, non return to estrus is an unreliable
procedure for pregnancy diagnosis in most domestic animal species.

Cocking of the tail

The pregnant female dromedary camels exhibit a characteristic behavior when approached
by a male or a person. The female assumes a stiffened posture with the head held high and tail
curled upwards. This is known as cocking of the tail. This behavior appears 14 to 15 days after
fertile mating and known to be 95% reliable for pregnancy diagnosis in quiet and calm dromedary
female camels. However, many false positives can be obtained in agitated females if the observer
is untrained. Tail cocking is also observed in the pregnant Bactrian camel although not with the
same intensity as in the dromedary female camel.

B) Udder development:

Besides the non-return to estrus a few of other visual signs of pregnancy appearing in late
pregnancy include increase in the size of the abdomen, development of the udder specially in dairy
heifers (4 months onwards), slight vaginal discharge (from 4-5 months onward in dairy cows) and
movements of the fetus visible externally (specially in fed cows on the right side of abdomen 6
months onwards). However, the accuracy of these visual diagnostic symptoms is always low and a
clinician must use them as a supplement to clinical diagnosis. As parturition approaches, the
mammary glands become grossly enlarged and oedematous and the teats take on a waxy, tumefied
appearance.

II. CLINICAL METHODS

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A). Recto-genital palpation/Trans-rectal/per-rectal Examination

Trans-rectal palpation is the oldest and most widely used method for early pregnancy
diagnosis in dairy cattle. In most large domestic animal species like cattle, buffaloes, mares and
female camels recto-genital palpation (with some limitations) is the easiest, cheapest and fastest
method of pregnancy diagnosis with little or nil harm to the animal and its fetus when performed
carefully. To a limited extent this method is used for pregnancy diagnosis in pigs. The method is
the technique of choice being taught to veterinary graduates and para-veterinary staff. Some of the
basic principles which a palpator must understand and the precautions that he must observe are
mentioned below and then the findings are described for cattle, buffaloes, mare, camels and pigs.
Trans-rectal palpation of the amniotic vesicle as an aid in determining pregnancy status in dairy
cattle, whereas slipping of the chorioallantoic membranes between the palpator‟s thumb and
forefinger starts between days 35-90.

Basic principle of rectal examination:

Examination of cows under epidural anaesthesia usually result in ballooning of rectum that
cannot be overcome until the anaesthesia wears off. The genital organs lie usually on the pelvic
floor during early pregnancy beneath the rectum in most species and in the abdominal cavity
during late gestation. The genital organs can thus be palpated indirectly by placing the hand in the
rectum evacuated of the faeces. The growth of the conceptus in either of the uterine horn leads to
sequential increase in the size, tenseness and palpable characteristics of the uterine cornua. Thus,
with experience the palpator can feel these changes in the uterus of a pregnant animal and with fair
to good accuracy predict pregnancy depending upon the species, stage of gestation and his
experience. Two bottle necks appear to be significant while performing rectal palpation: i) the
peristalsis that occurs in the rectal musculature, which produces obstacles in palpation and ii)
ballooning of the rectal wall due to entry of air inside.

The palpator must stop making movements of arm during a peristaltic wave (while still
keeping his hand inside the rectum) wait for 1-2 minutes and then start palpation again when the
peristalsis has subsided. The ballooning of rectum can be easily appreciated, by the finding that
the operator can move his hand up and down in the rectum without resistance when the rectum is
ballooned. The operator must move his hand back and forth (known as back racking) to evacuate
the faeces. The faeces must be removed without taking out the hand completely.

After proper restraint and wearing of proper clothing and also proper lubrication, the operator
must make a cone of his hand and push it inside the rectum. Sufficient lubrication must be used
while introducing the hand in the rectum. Non-irritating soap and water or liquid paraffin is a
suitable lubricant. Since, the faeces of the mare are harder and the peristalsis stronger, more
frequent lubrication is essential in the mare. Tail bandaging is also advantageous in the mare to
avoid tail hairs enter the rectum and cause damage to the rectal wall. The anal sphincter dilates
and the hand enters inside the rectum. The cervix which is a hard round to oval or sometimes
caudally enlarged disfigured structure is the land mark for location of genital structures in cattle
and buffaloes. This can be located by sliding the hand in an arc like fashion from dorsal to ventral
side. The cervix is followed further to locate the uterine body and the uterine horns. These
structures can be pulled caudally when located at the pelvic brim or further, by retracting the broad
ligament or hooking the inter-cornual ligament by the index finger. When the pregnancy is beyond
 P a g e | 51

60 days this cannot usually be done and the operator has to move his hand further in the rectum, so
as to locate the intra-abdominally placed uterus and palpate other features diagnostic of
pregnancy.
Rectal palpation is the most widely used method and it is non invasive, accurate and results
are quickly known. During rectal palpation cervix and uterus and its contents are palpated and up
to 120 days of pregnancy ovaries can also be palpated. In herd where AI is used, the first palpation
should be made between 35-42 days of insemination. Even though, palpation at this stage require
a greater expertise, the main interest should be to identify those cows which are non pregnant so
that they can be observed more carefully.
In the mare, the cervix is not easily palpable and hence the ovaries are the land mark for
rectal palpation especially for novice palpators. They are located about 10-20 cm cranial to the
shaft of the ilium bone and about 5 to 10 cm below the lumbar vertebrae in non-pregnant mares,
and in mare during early pregnancy. After locating one ovary the hand is passed down the utero-
ovarian ligament to locate the uterus.

Female camels are examined in a sitting position with both hind, and forelegs tied together
separately with ropes. An assistant holds the head tightly. Vicious females often require pressure
on the back by legs of persons standing on both sides to prevent side wise movements. A striking
feature of the genitalia of the female camel is the shortness of the right uterine horn. This is
probably, because of the existence of exclusive left horn pregnancies in this species. Recto-genital
palpation is similar to that described for cattle and buffalo as the cervix is easily palpable.
Rectal palpation is only possible to a limited extent in the large sized sows. It is barely
possible in gilts and in small sized breeds of sows.

PRECAUTIONS DURING RECTAL PALPATION

When performed gently and carefully, rectal palpation is a non-invasive procedure. The
following points would be helpful in minimizing damage to the animal and the examiner as well.
1) Ruthless movements of the hand in the rectum should be avoided. Avoid palpations during
a peristaltic wave.
2) Examiners must trim their nails and avoid using dirty soiled sleeves.
3) Rectal examination without a sleeve must be avoided specially in mares to avoid
contracting diseases and obnoxious odours. Sleeves must be replaced after examination of
2-5 animals, or better after each examination.
4) Rectal palpation of an animal suffering from fever should be extremely gentle or better
avoided as the blood vessels are more fragile and bleed easily. Similarly examining an
animal with rectal tear or rectal fistula is hazardous. Whenever, a clinician notices such
conditions he must bring them to the notice of the owner or else he would blame the
clinician.
5) Compared to cattle rectal palpation in buffaloes must be gentle as the rectal mucosa is
more fragile and bleed easily.
6) Clinicians must assure that even if the animal kicks it does not harm them, and so also the
palpators must also be cautious that sudden sideways movement of the animal with the
operators hand inside can cause fracture of the operators arm and hence due care must be
exercised.
 P a g e | 52

7) Uncareful palpation of the uterine horns with undue pressure can cause rupture of the
amniotic vesicle and loss of an early pregnancy (iatrogenic embryonic mortality) and hence
this must be avoided.

Palpable findings of pregnancy in cattle and buffaloes

Two things must be kept in mind by clinicians in making positive diagnosis of pregnancy
by rectal palpations in cattle and buffalo. The first is, that when the palpator in unable to detect
any of the palpable characteristics mentioned herein, he must neither comment positively or
negatively as both would be frustrating both to the clinician and the owner on a later date. He must
better admit the fact that he is not able to detect out properly and the animal must be re-submitted
for examination 15-30 days later preferably after a fasting. The second thing that clinicians must
keep in mind is the accuracy of the gestation period (this is especially applicable for the 5-8 month
period in cattle and 5-9 months in buffalo). An approximation of the gestation period must be
conveyed to the owner rather than an accurate period. Although experienced clinicians can more
precisely comment on the gestation period after an examination it is usually safe to be
approximate. When pregnancy examinations are made early (Day 30-45) the possibility of an
early embryonic death must be kept in mind and a confirmation of pregnancy must be done only
after re-examination at a later period (60-90 days).

Owners often consult veterinarians on the fetal viability during mid to late gestation. It is
often difficult to comment positively by a single rectal palpation. Until unless characteristic
changes in the uterus and fetus are palpable negative comments must better be avoided. The usual
test for fetal viability during this period is the movement of the fetus in response to stimuli by the
examiners hand (movement of a fetal leg when pressed by hand or suckling movements by the calf
when a finger is touched in the mouth) however; this may be sometimes misleading especially
when the fetus is depressed.
It is likely that the rectal examination of cows from 42 days of gestation is safe and reliable
method when preferred carefully and skilfully.
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Location of pregnant uterus

The pregnant uterine horn under goes sequential changes in size, location and morphology
and they form the basis for pregnancy diagnosis. The early pregnant uterus lies in the pelvic cavity
in heifers, and just ahead of the pelvic brim in pleuriparous large sized cows. As it grows in size,
its growth is forward so it starts descending into the abdominal cavity (approx 3½ - 4 months). At
approximately 4½ - 5 months it reaches the abdominal floor and at this time only cervix is
palpable within the pelvic cavity which is also drawn forward. The growth is then forward and
then again upwards. The entire uterus or the fetus is therefore barely palpable during the 4 – 6½
months period and diagnosis has to be dependent on other features of pregnancy (placentomes or
fremitus). After this period the fetal parts are usually palpable and clinicians find no difficulty in
commenting whether the animal is pregnant or non-pregnant. During early pregnancy (day 30-60)
clinicians have to depend on finding of the fetal membrane slip or the palpation of the amniotic
vesicle.

The definite signs of pregnancy in the cow as determined by rectal palpation are i)
palpation of enlarged uterine horn containing the placental fluids ii) palpation of the amniotic
vesicle iii) slipping of the fetal membranes iv) palpation or ballottement of the fetus v) Palpation
of the placentomes vi) palpation of enlarged thin walled “whirring” middle uterine arteries.
 P a g e | 54

Uterine changes

The increase in the diameter of the uterine horns is characterized by a thinning of the
uterine wall and the feeling of a fluid filled structure. Unilateral enlargement of uterine horn and
disparity in size, thinning of the uterine wall, fluid filled fluctuation of enlarged horn appears as
early as on day 35. By 40-90 days of pregnancy, the uterus feels like a thick rubber balloon
nearly filled with water. Around 90 days, tapping of distended horn with fingers may reveal the
fetus rather like a piece of wood floating in the fluid beneath. The volume of fluid increases
rapidly the first 5 months of pregnancy and then increase slowly. With advancing pregnancy the
non pregnant horn may also increase in size slightly.

The amniotic vesicle

The amniotic vesicle can be palpated with due care between 30-65 days of gestation as a
movable oval object within the uterine lumen, many a times at the apex of the cornua. The vesicle
is turgid, early in pregnancy but becomes flaccid with advancing gestation until days 65-70 when
it is difficult to detect at all.

Slipping of the fetal membranes

The fetal membrane slip can be felt between 35-90 days of gestation. The entire uterine
horn must be grasped in the palm and allowed to slip while the fingers compress it gently. The
allantois-chorion, slip between the thumb and fingers before the uterine wall escapes. It is felt like
a connective tissue band. The pressure must be gentle. The entire diameter of each uterine horn
must be palpated.
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Palpation of placentomes

The presence of placentomes is another positive sign of pregnancy. Placentomes are

formed by 60-70 days of pregnancy and they can be detected from about 75-80 days to term. The
period of pregnancy when the uterus has descended into the abdominal cavity and the fetus is not
palpable, palpation of a placentome is the surest indication that the cow/buffalo is pregnant. Since
there is great variation in size among individual placentomes (those nearest the fetus are the
largest), their usefulness in aging a pregnancy is limited. In general, they can be detected as soft,
thickened lumps in the uterine wall and are more easily detected as pregnancy advances.

Palpation of the fetus

The palpation of the fetus itself is a positive sign of pregnancy. Depending on the skill of
the examiner and the location of the fetus, the fetus can be palpated from the time of amniotic
softening (65 to 70 days) to term. However, in large sized cows the abdomen should be lifted up
by a bamboo held by two attendants on either side of the abdomen to palpate a fetus during mid
gestation (4½ - 6½ months). The whole of the fetus is palpable many a times only during early
gestation (2 to 4 months). The size of the fetus is approximately that of a mouse or rat at 2 and 3
months and it increases to the size of a small cat at 4 months, a large cat at 5 months and a beagle
dog at 6 months respectively. The maximum fetal growth occurs during the last one to one and a
half month of gestation and estimates of predicting pregnancy status depend upon the experience
of the clinician and location of the fetal parts. Beyond 8 months of gestation, fetal parts (legs,
head) are palpable within the pelvic cavity or just cranial to the pelvic brim. Palpation of a fetal
extremity is sufficient evidence for pregnancy if other uterine findings are normal.

Palpation of middle uterine arteries (fremitus)

The major supply of blood for the gravid uterus arrives via the middle uterine artery, which
enlarge considerably as pregnancy progresses. The artery runs in the broad ligament along a
tortuous course, passing downwards, forwards and towards the midline over the pelvic brim close
to the junction between pubis and ilium. Usually, it is identified 5-10 cm lateral to cervix. They
can thus, be felt by the hand directed laterally towards the iliac shaft. Enlargement of the uterine
artery ipsilateral to the pregnant horn is detectable after 85 days of gestation. By approximately
120 days, the blood flow within the artery increases to a point where the blood flow is palpable as
a buzzing sensation, also called “thrill” or “fremitus”. By 7 to 8 months the fremitus is often
palpable on the side of the non-pregnant uterine horn also. The detection of fremitus is a positive
sign of pregnancy. If the fremitus was felt in early pregnancy and then disappear indicates death of
fetus.

Palpation of ovaries

As the pregnancy advances ovaries may be dragged forward along with the pregnant uterus
and may not be palpable beyond the four to five months.
 P a g e | 56

Vaginal changes

During pregnancy the vagina usually develops a pale, dry sticky mucus membrane. The
cervix is closed and the cervical (mucus) seal covers the external os by day 40 to 120 of
pregnancy. Slight degree of vaginal discharge is evident in some cows beyond 5 months of
pregnancy but the cervical seal liquefies only prior to parturition or abortion and is discharged in
strings.

DIFFERENTIAL DIAGNOSIS OF PREGNANCY IN BOVINES

Owners are many times perplexed on the outcome of a pregnancy that did not progress
normally and often present their animals for a definitive diagnosis to a clinician. Some
pathological conditions mentioned in detail in subsequent chapters often should be identified to be
different from a normal pregnancy. The palpable differences of these conditions are briefly
described below.

a). Mummified fetus

A uterus with mummified fetus has thick uterine walls, absence of fluid and placentomes
and a hard palpable structure. The fetus is closely opposing the uterine wall. The fremitus is
absent.

b). Maceration of the fetus

There is copious vaginal pus discharge. The uterine wall is thick and doughy. There is no
dorsal bulging of the uterus and placentomes are not palpable. Parts of bones are sometimes
palpable separately floating tacked up. The fremitus is absent.

c). Pyometra/Mucometra

These two conditions are many times difficult to differentiate from normal pregnancy
especially when the pus or mucus is present in enormous quantity (sometimes 20-40 litres) so that
the uterus is largely enlarged and placed on the abdominal floor. In pyometra the uterine wall is
thick, uterus is doughy and placentomes or fetus is not palpable and the fremitus is absent.
However, it is always safer to make a re-examination 1 to 2 months later when the clinician is not
sure, or make use of ultrasonography to differentiate the condition. Sonographic evaluation would
depict anechogenic fluid without cotyledons and echogenic material accumulated. A clinical
therapy to terminate the corpus luteum on an assumption that the condition is pyometra could be
hazardous and loss to the owner, if unfortunately the condition was a normal pregnancy. In
mucometra, the positive findings of pregnancy are absent, but, contrary to pyometra the uterine
wall is thin. Ultrasonography can easily differentiate the condition from pregnancy. When these
two conditions are to be differentiated from early pregnancy a characteristic feature found most
often is the bilateral enlargement of both uterine horns, which is not found in pregnancy. The fetus
is not palpable in these two pathologies, and often there is a history of vaginal discharge.
 P a g e | 57

d). Urinary Bladder

Infrequently, full of urinary bladder creates confusion for the presence of an early
pregnancy (60-90 days) in bovines. This can be easily differentiated by the absence of palpation of
both uterine horns and the ease with which the animal urinates when the bladder is gently
massaged leading to disappearance of the enlargement

e). Rumen
Rumen feels doughy consistency, whereas gravid uterus gives a live fluid feeling
sensation.

f).Tumors
Rarely tumors of the genital tract can create confusion, but their consistency and location
is different.
PALPABLE FINDINGS OF PREGNANCY IN MARES

The early diagnosis of pregnancy in the mare has its own significance. A limited breeding
season in some breed registries (for e.g. Thoroughbred) warrants that the non pregnant mares are
diagnosed early and steps be taken to breed the mare again in the same breeding season. Some
peculiarities of the equine pregnancy are that an ovarian source of progesterone is essential for
maintenance of pregnancy until approximately days 50 to 70; thereafter, the fetal-placental unit
begins producing sufficient progestagens to support pregnancy.

The early equine feto-maternal axis secretes sufficient estrogen and the equine embryo
stabilizes by day 16. The endometrial cups form at about 35 days of gestation and remain up to
150 days. Secondary, accessory and supplemental corpora lutea (day 40) form on the mare ovary
and all corpora lutea regress by day 180-210.

Diagnosis of an early pregnancy by rectal palpation is one of the easiest and accurate
means for experienced personnel. However, a diagnosis at day 18-20 should be confirmed at day
45-60 because of a late implantation of the equine embryo and chances of early embryonic death
and reabsorption. The basis of pregnancy diagnosis in the equine species lies on the changes that
follow in the cervix, uterus and ovaries. Placentomes are not found in the equine species and the
fremitus or fetal membrane slip is not marked, therefore some of the parameters used in cattle
cannot be used in the equine species directly.

Cervix

As early as 16 to 18 days after ovulation, the cervix of the pregnant mare becomes tightly
closed, firm, slender, and elongated. Between 16 to 30 days with the experience the cervix can be
palpated on the floor of pelvis as a rigid firm structure.

Uterus
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Most pregnancy diagnosis in the mare by rectal palpation is done by palpation of the
uterus. The parameters used for a positive diagnosis are as follows:

a). Uterine tone and thickness

One of the positive sign that a mare has conceived to a breeding is the finding of increase
in uterine wall thickness and a marked tone. The endometrial folds are no longer palpable as folds
of tissue. The uterus becomes tubular, smooth and firm. The uterine tone appears at days 15-16
and continues up to day 48 to 55. This is diagnostic except in cases of endometritis or in mares
bred at the foal heat, which are still undergoing uterine involution.

b). Uterine size and embryonic vesicle

The conceptus becomes positioned at the base of one of the uterine horns at the junction
between uterine horn and body. At 25-28 days, a small bulge may be palpable in maiden mares.
The bulge progressively increases in size and is palpable in most mares by day 30-35 (approx 3-4
cm in diameter). A small notch can be readily appreciated on both sides of the bulge. By day 42-
45, the conceptus occupies approximately half of the gravid horn and is 5 to 7 cm in diameter. The
uterine wall over the bulge in thin.

At 60 days of gestation, nearly the entire gravid horn and half of the uterine body are filled
with the conceptus but the non-gravid horn remains small. The pregnancy is like an elongated
football and is nearly similar to a 60 day pregnancy in cattle. The tonicity is markedly reduced at
this time. The 60 day conceptus is approximately 8-10 cm in diameter and 12 to 15 cm in length.

c). Palpation of fetus

The fetus is active after 40 days and mobile after 70 days. Palpation of the turgidity of the
conceptus is absent by day 90, and the fetus is palpable, which feels like a small, heavy,
submerged but floating object as the hand contacts it. In most mares it is usually possible to
palpate the fetus per rectum from the third month onward throughout the gestation. In a few deep
bodied and large sized mares palpation of the fetus is difficult from the fifth to seventh months of
gestation. In these mares the location of uterus and ovaries would aid the diagnosis.

d). Location of the uterus

The uterus is located in the pelvic cavity or just at the pelvic brim until day 90. At 100-200
days, the gravid uterus is positioned cranial to the pelvic brim in the abdominal cavity. The
ovaries are positioned cranial and ventral to their normal positions and closer together. By 5 to 7
months the uterus is positioned low in the abdomen and it is difficult to thoroughly evaluate the
conceptus by palpation per rectum. During the sixth and seventh months, the horns are
approximately perpendicular to the dorsal cranial aspect of the uterine body. Beyond the seven to
eight months the fetus is easily palpable by rectal palpation.

e). Ovarian palpation

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Both ovaries should be palpated. Both ovaries usually are enlarged from 18 to 40 days as a
result of follicular development and the CL is not palpable. From 40 to 120 days, extensive
ovarian activity with ovulations, luteinization, and development of secondary corpora lutea is
evident. Follicular activity decreases from 120 days to term, and the ovaries become small and
inactive. The position of the ovaries up to 60 days of pregnancy is similar to that for the non-
pregnant mare. From then on, they are drawn cranially and medially but remain dorsal to the
uterus. The finding of both ovaries nearer to each other and close to the pelvic floor is a positive
indication for pregnancy when the uterus or other structures are difficult to palpate (3 to 5
months). From 5 months of pregnancy onwards, the ovaries usually are not palpable as they are
under the broad ligaments.

RECTAL PALPATION IN THE SOW

To a limited extent rectal palpation has been described as a method of pregnancy diagnosis
in the sow. Sows are examined while standing in gestation crates or pens. This technique is based
on examination of the cervix and uterus, together with palpation of the middle uterine artery to
assess size, degree of tone, and type of pulse. At around 21-30 days of gestation the bifurcation of
the cornua is less distinct, the cervix and uterine walls are flaccid and thin. The middle uterine
artery is 5-8 mm in diameter and more easily identified. The uterus becomes progressively thin
walled and ill defined by 31-60 days and fremitus can be identified at 37 days. Beyond 60 days the
fremitus is very strong, however, piglets can only be palpated towards the end of gestation. The
procedure is often difficult in gilts and small sized breeds because of a small pelvis and too small
rectum. False positive diagnosis is likely if the external iliac artery is mistakenly identified as the
middle uterine artery. The technique is however, not popular at most locations.

RECTAL PALPATION IN FEMALE CAMELS

The corpus luteum formed on the camel ovary (ovulation is induced by mating) persists
and is necessary for the entire gestation. The persistence of the CL is one of the earliest sign of
pregnancy as otherwise; the luteal phase is very short. The CL is out of reach by day 90 of
pregnancy. The left uterine horn is inherently longer than the right horn and this must be kept in
mind when making pregnancy diagnosis in female camels. The earliest detection of uterine change
(increase in diameter and appearance of fluctuation) is palpable at about 40-15 days. Between 60-
70 days, the left uterine horn is increased about twice to its non-gravid size, has a thin wall and
fluctuates.

The uterus becomes cranial and ventral after the third month of pregnancy. The cervix is
pulled forward and lies just at the pelvic brim by 4 months. At 5 months of pregnancy, the uterus
is completely in an abdominal position with a small degree of fluctuation but the fetus is not
always palpable. The fetus becomes palpable again beyond the 6th month of pregnancy, first with
ballottement, and then, the head and legs become easily palpable by the 7-8 months as the fetus
starts its ascent. By the 9th month, movement can be observed by inspection of the right flank of
 P a g e | 60

the animal. At around 11 months the fetal legs can be easily found in the pelvic cavity. The precise
estimation of the stage of pregnancy beyond 3 months, because of the absence of cotyledons and
difficulty in reaching the fetus in this species is difficult. The fetal membrane slip is not seen in
camels because of a diffuse placenta.

B). Ultrasonography

During the last couple of years, ultrasonography has gained popularity in veterinary
medicine and has become the method of choice for diagnostic imaging of the various organs of the
body, including reproductive organs. Ultrasound is a high frequency sound wave. Sounds audible
to the human ear vary between 20 to 20,000 Hertz (Hz) (Cycles per second) while ultrasound
waves are of frequency higher than this, and for most diagnostic applications frequencies of 1-10
MHz are used. Even the short distance between the transducer (which emits and receives
ultrasound signals) and the patient must be bridged by a suitable coupling gel.

Basic principle

The ultrasound equipment basically consists of a transducer and a scan converter. The
transducer is the ultrasound producing part. It is fitted with a piezoelectric crystal (Lead–zirconate
– titanate or others) which when stimulated by a high voltage current emits the ultrasound. The
ultrasound is transmitted to the patient from the transducer and propagates through the tissues. The
ultrasound beam is either reflected back, partially absorbed or entirely absorbed. The returning
beam (echoes) meets back and deforms the crystals in the transducer. This mechanical energy is
converted back to an electrical signal proportional to the strength of the echo and delayed by a
time roughly proportional to the distance travelled. The scan converter interprets the variations in
brightness displayed on the cathode ray tube of a B-mode system (or as a variation in amplitude in
A-mode oscilloscope screen) and also stores images when required. The ultrasound is emitted in a
pulse – echo manner. A pulse of ultrasound is emitted and its reflection perceived prior to
emission of the next pulse.

Types of instruments and some definitions

For most diagnostic veterinary purposes B-mode, real time ultrasonography is used
employing different types of transducers. Transducers used commonly in veterinary reproductive
practice are the linear trans-rectal transducer (frequencies of 5-10 MHz) and the sector
transabdominal transducer (frequencies of 1-4.0 MHz). For most reproductive diagnostic work,
linear array trans-rectal transducers are employed in cattle, buffaloes, mares and female camels.
Small sized trans-rectal transducers are also used for early pregnancy diagnosis in small ruminants
(sheep and goat).

For bitches mostly transabdominal sector transducers are useful for pregnancy diagnosis
with frequencies from 2 to 4.0 MHz. The same transducers can be used for pregnancy diagnosis in
sheep and goat beyond day 40 of gestation. However, in order to visualize an early pregnancy or
the non pregnant bitch uterus trans-rectal transducers of high frequency (5-7.5 or 10.0 MHz) are
essential.
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A wide variety of transducers with single, dual, or multiple frequency and multiple
functions are available and clinicians must decide what type of instrument and the transducer he
must purchase depending upon his work. Advancement to ultrasonography includes diagnostic
imaging using color Doppler and magnetic resonance imaging.

When performing ultrasonography it is important for the sonographer to have basic

knowledge of the anatomic location of the different organs to be visualized and problems that can
be encountered in obtaining and interpreting the images obtained. A few of the common terms
related to ultrasonography are described below:-
Anechoic (echolucent): In absence of any echo, the images seen on screen appear black. It is
presented by complete transmission of sound waves such as through liquids like follicular fluid,
chorionic or amniotic fluid.
Hyperechoic: It represents the bright echoes which appear as white on screen. Such images are
given by highly reflective interfaces or dense tissues such as fetal bones, bovine cervix etc.
Hypoechoic: These appear as grey images on dark screen and are given by interfaces of moderate
reflection such as soft tissues.

A-mode (Amplitude modulation) or 2D: A one-element (one dimensional) display with

time (distance) on the horizontal axis.

B-Mode (Brightness modulation): A compound A-mode scan with amplitude translated

into a brightness scale. Location on the display is related to position and depth. When an
ultrasound beam meets a moving object the reflected ultrasound is either of increased or decreased
frequency, depending upon whether the motion is towards or away from the transducer.

M-Mode (Motion mode): A rapidly updated one dimensional B-mode display with time on
the second axis to allow study of moving structures. It is used in cardiology.

Echogenic: A structure causing a marked reflection of the ultrasound beam. A change in

echogenicity in a homogeneous structure may indicate a pathological change.

Trans-rectal ultrasonography

The animal to be examined is properly restrained, the faeces are evacuated from the rectum
and the perineum washed with water. The coupling gel is applied to cover the transducer. The
operator keeps the transducer in his gloved arm and takes it inside the rectum. The uterine horn on
one side is scanned to the entire length and the ovary of that side is also scanned. The operator
then moves his hand to the other uterine horn and ovaries. If the pregnancy is advanced the
operator may have to take his hand deeper. When required the images seen may be frozen and the
diameter of the structures measured by inbuilt callipers with the machine. At 60 days the fetal
crown length is approximately 6 cm. The amount of fluid and thickness can also be measured. The
transducer is then taken out and the perineum is washed again.
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Trans-abdominal (Trans-cutaneous) ultrasonography

For reproductive trans-abdominal ultrasonography in sheep and goats the hair must be
clipped from just above the udder and 15 to 20 cm ahead of the udder on both sides of the
abdomen. The transducer is placed above the udder between the thigh and abdomen preferably the
left side and moved in a „W‟ shape from one side of the abdomen to the other side. The procedure
can be performed with the animal standing (sector scan) or in lateral or ventral recumbency (linear
scan) depending upon the type of transducer being used.

For pregnancy diagnosis, sows are examined in a standing position when using sector
scanners (3.5-5.0 MH2 frequency) or Doppler instruments (which are more frequently used in pigs
to detect fetal heart beat, fetal movements and uterine artery pulsations). The transducers are
placed over the abdomen just medial to the stifle skin fold, just at the level of second last teat.

Trans-abdominal imaging of the uterus of a bitch can be done with the bitch in the standing
position after clipping the hair of the ventral abdomen. However, dorsal and lateral recumbent
positions may also be used. The uterus lies dorsal to the bladder, but its position may vary with the
extent of bladder filling, and the size of the uterus. During early pregnancy the uterus has a more
dorsal position in the abdomen of a standing bitch, but with advanced pregnancy the uterus is
closer to the ventral lower abdominal wall.

Sonographic findings during pregnancy

When the ultrasound strikes to a smooth and wide structure (e.g. a CL) there will be almost
total return of echoes where the sound waves strike at right angles, giving an intensified signal that
appears on the screen as a whiter shade of grey. This is referred to as specular reflection, and is
often seen in early pregnancy when imaging the embryonic vesicle.

With these basic things in mind one has also to keep in mind that fluid filled structures
appear black (anechoic), hard structures (like the bone) appear white (hyperechoic) and other
structures with their structure midway between the bone and fluid appear grey (hypoechoic). The
basic diagnosis of pregnancy lies in the identification of structures from black, grey or white scale.

The earliest sonographic finding for pregnancy in most animals is the appearance of
anechoic fluid within the uterine lumen. This fluid goes on increasing to a stage when the embryo
proper becomes visible as a hypoechoic structure floating within this fluid and progressively the
fetal structures become more clearly visible along with the fetal membranes. The viability of a
growing fetus is ascertained when the fetal heart beat becomes visible as a hypoechoic flickering
structure.

With different probes the amniotic vesicle/fetal fluid is visible in most species between
days 18-22 except in the mare in which it appears earlier (day 10-16). The fetal heart beat can be
seen between day 24-30 and the fetus itself between days 25-30 in most species. The cotyledons
are visible between day 30-40 and fetal extremity/bone by day 57-60 in cattle and day 70 in sheep.
 P a g e | 63

It therefore, appears that the fetal fluid, fetal heart beat and the fetus become sonographically
visible at nearly the same time in most species. The most appropriate time for pregnancy diagnosis
using ultrasonography with high accuracy in cattle, camel and buffaloes appears to be day 28-30
using a trans-rectal linear array probe of 5.0 to 7.5 MHz frequencies. Using the same probe
pregnancy can be diagnosed with sufficient accuracy a little earlier (24-25 days) in the mare.
Under field conditions a transabdominal probe (linear or sector 3.5 to 5.0 MHz frequency) is
generally used and this can diagnose pregnancy earliest at day 40-50 in sheep and goats with
reasonable accuracy as the fluid and cotyledons are easily visible sonographically at this time. The
diagnosis of pregnancy in the bitch requires a transabdominal probe (of frequency 3.5 to 5.0 MHz)
to visualize pregnancy with accuracy from 25-30 days. The visualization of earlier pregnancy or
the visualization of a non-pregnant bitch, uterus necessitates the use of probes of higher frequency
(7.5 to 10.0 MHz) as the uterus lie closer to the skin. Doppler ultrasonography and A-mode probes
are generally used in pigs for pregnancy diagnosis but B-mode probes with frequencies of 3.5 to
5.00 MHz can diagnose pregnancy with high accuracy between days 25-30 post mating.

C). RADIOGRAPHY

To a limited extent radiography has been used for pregnancy diagnosis in the small
ruminants (sheep and goat), the companion animals (dog and cat) and rarely in pigs. The technique
is known to be good in evaluating fetal numbers in the bitch and cat, but is poor in evaluating fetal
viability. Moreover, the high cost and the hazards of exposure to growing fetuses to x-rays limit
the use of radiography as a routine procedure, and warrant its use in specialized cases. Mostly, a
single radiograph taken with the animal in lateral recumbency is sufficient however; sometimes a
dorsal or a dorso-ventral view may be required. Radiography is suggested to be done only after
day 90 in sheep and goat.
The fetal skeletons begin to calcify only after the sixth week in sows and hence
radiography should be performed only after this time for pregnancy diagnosis in sows. Because
pregnancy can be diagnosed with high accuracy with other methods, radiography is seldom
performed for pregnancy diagnosis in sows.

D). ABDOMINAL BALLOTTEMENT

Abdominal palpation and abdominal ballottement of the fetus is possible to some extent in
bovines during late gestation (7 months onward). In cattle abdominal ballottement is performed by
placing the fist over the lower right abdominal wall and pushing it in an intermittent manner in a
dorsal medial direction deeply. This is usually possible in lean cows after the 7th month of
gestation. The fetus can be felt as a hard solid object floating in fluid. Palpation of fetuses
through the abdomen is possible in sheep and goat only beyond 4 months of pregnancy by lifting
the abdomen held between both hands and location of bony fetal structures.
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E). LAPAROSCOPY

Laparoscopy can be used as a method of pregnancy diagnosis by directly visualizing the

genitalia in animals however, the invasive nature of the technique, the high cost of equipment and
clinic required, and the availability of non invasive techniques limits the use of this technique as a
means of pregnancy diagnosis in most animals.

III. LABORATORY METHODS FOR PREGNANCY DIAGNOSIS

A). Vaginal Biopsy

Histological assessment of the number of layers of the stratified squamous epithelium of

the vaginal mucosa obtained by biopsy can be used as a method of diagnosing pregnancy in the
sow and to a limited extent in sheep. The accuracy of this method between 18 and 22 days is 97%
and 94% for the diagnosis of pregnancy and non-pregnancy respectively in the sow. The basis for
the test is the decrease in the layers of the stratum germinatum (vaginal epithelium cells: to 3 to 4
layers at 18-25 days of pregnancy) under the influence of progesterone. The number of layers is
high at estrus (around 20 layers) due to influence of estrogen hormone. The limitations of such a
diagnosis are the invasive nature of the test, poor results due to improper sampling and improper
tissue processing.

B). IMMUNOLOGICAL METHODS:

i. Progesterone hormone assay:

The corpus luteum formed on the ovary subsequent to ovulation produces progesterone for
maintenance of pregnancy for a reasonable time period in some species and for entire gestation in
other species like the cow, buffalo, goat and sow. In normally cycling cows and buffaloes, the
CL is lysed because of the effects of prostaglandins from the uterus if the animal is not pregnant,
and thus the progesterone level goes down. Therefore, low progesterone concentrations in
maternal blood at 18 to 24 days post breeding can predict that the animal is non-pregnant and high
progesterone gives an insight that probably the animal is pregnant. The specificity of progesterone
tests conducted between 18 and 24 days post breeding have shown a specificity of around 98%
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and is the easiest proven method for identifying non-pregnant (regularly cycling) animals post
breeding. However, for the pregnant animals the accuracy of the test is low (75%) because of early
embryonic death which alter the results. Commercially available ELISA, plasma or milk
progesterone assay kits have not become popular due to their high cost and a low specificity. Non
pregnant cows not returning to estrus and pregnant cows in which embryonic death occurs at a
later time can both give false results. Likewise, in mares, sheep, goats, buffaloes, camels and sows
assay of plasma or milk progesterone is not very accurate for diagnosis of pregnancy.
Since progesterone concentration in the peripheral blood of pregnant bitches is similar to
those of non pregnant bitches, and since there is no placental progesterone produced in the
pregnant bitch hence progesterone assay cannot be used to diagnose pregnancy in the bitch.

ii). Oestrone sulphate:

The estrone sulfate is produced by the feto-maternal axis or the conceptus and therefore its
presence in urine, milk, faeces or blood is an indicator of pregnancy. The main oestrogens in the
mare are oestrone and a ketonic steroid, equilin. However, oestradiol-17β, oestradiol-17α and
equilenin are also present. The identification of Oestrone sulphate in the milk of a cow at 105 days
of gestation or later is a very reliable method of pregnancy diagnosis. The appropriate day, at
which detection of estrone sulphate detection is possible in the body fluids or secretions are
mentioned in Table 1.

Table 1: Appearance of estrogen in domestic animals during pregnancy

Species Day of detection
Mare Maternal estrogen high after 60 days, conjugated
urinary estrogens high after 150 days
Cow, Day 105 of gestation
Goats High after day 50 of gestation
Sows Rise start at 20 days peak at 25-30 days followed by a
decline at 45 days and again a rise at 70-80 days to term
Slightly increased at implantation and remain constantly
Bitch high for rest of gestation and decline 2 days pre-partum
Detectable by day 70; rise thereafter till 2 days pre-
Sheep partum
Buffalo Appear at day 150 of gestation in the serum
Camel Increase start at day 50 and peak from day 90-300

The detection of estrogens depends on the availability of suitable laboratory and

availability of commercial assay kits. Laboratories evaluating concentrations of estrogens in urine
or serum usually are equipped with radioimmunoassay, enzyme immuno-assay or other more
precise and specific diagnostic modalities for assay of steroids in urine, serum, faeces or other
body fluids. Evaluation of steroids like estrogen from faeces is especially helpful for zoo and feral
species where faeces are the most easily collected specimens. Commercial kits have been
developed for pregnancy detection in mares by using on farm kits like Wee-Foal-Checker® or
Equitest ES® which require urine or serum as the test material. These commercially available tests
are recommended to be performed only after 120 days of gestation and specially suggested for
miniature horses and donkeys in which pregnancy diagnosis by rectal palpation or
ultrasonography is extremely difficult.
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Cuboni test: To detect urinary estrogen

This test was first developed by Cuboni (1934) and modified later (Galina and Cox, 1969).
The test is performed in the mare for detection of pregnancy through assay of urinary conjugated
estrogens. The Cuboni test is only effective between 150 and 300 days of gestation, and also
predicts fetal viability.

The test is preformed as follows:

“To 15 ml of urine 3 ml of concentrated hydrochloric acid is added and heated in a water

bath for 10 minutes and then cooled under a tap. To this 18 ml of benzene is added and shaken
vigorously for half a minute. The supernatant (mainly benzene) is collected in another tube and 3
to 10 ml of concentrated sulphuric acid is added and the mixture heated in a water bath at 80°C for
5 minutes, and then allowed to cool. A positive (pregnant) test is indicated by the appearance of a
dark, only green fluorescent color in the lower sulphuric acid layer and a negative (non-pregnant)
is characterized by absence of fluorescent color and presence of a brownish color.”

Other chemical tests previously described for the detection of urinary estrogens in mares
include the vaginal cornification, mouse or rat tests using ovariectomised female rats or mice, and
the phenolsulphonic acid test. In the mouse test, the serum or urine from pregnant mares when
injected to ovariectomised mouse or rats would induce vaginal edema, appearance of cornified
cells and mucus discharge due to presence of estrogens in the pregnant mare‟s serum or urine.

The phenolsulphonic acid test utilizes ether extraction of urinary estrogens subsequent to
removal of urinary pigments by hydrolysis and their conjugation with phenolsulphonic acid
reagent after evaporation of the ether. The final reaction gives a pink to cherry red color if the
urine is from pregnant mares. The test is lengthy and cumbersome and requires special precaution
in processing which has turned out this test of historic importance only. These tests are 70-80%
accurate when performed between the 120 to 250 days of pregnancy.

iii). PMSG(Pregnant Mare serum gonadotrophin)/eCG(Equine chorionic

gonadotrophin) : Endometrial cups are formed as early as day 35 and secrete the equine
chorionic gonadotrophin (eCG) which appear in blood of pregnant mare as early as on day 38-42
and can be detected be detected from serum of pregnant equine females. The eCG continues to be
secreted from day 40 to 140 days of pregnancy and disappear after day 150. Once the endometrial
cups are formed, they will persist and secrete eCG even if the fetus has died. They regress at the
time that they would have done if the pregnancy had continued normally.

On farm testing: National Research Centre on Equines (NRCE), Hisar has developed a kit
named as Pregmare Kit which detects eCG in serum samples of pregnant mare between day 35-
125 post covering. The kit requires 5 drops of blood collected from the muzzle by using the lancet
provided with the kit. This color change test requires about one hour completing.
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iv). Early pregnancy/conception factor

This protein molecule was first identified in pregnant mice by Morton et al.(1987) and
later in sheep and cattle by Nancarrow et al. (1981) by using the rosette inhibition bioassay. An
immunosuppressive early pregnancy factor (EPF) appears as early as 6 to 48 h of mating which
functions to suppress the maternal immune response thereby allowing for pregnancy to proceed. In
cattle significant differences in rosette inhibition titer are observed between pregnant and non
pregnant cows on day 13-16 and 25 post breeding suggesting that measurement of EPF activity
may be useful as an indirect method of pregnancy diagnosis. In cow EPF has a molecular weight
of 200000. Commercially available test kits are available that use the dipstick principle and can
detect ECF in serum and milk from as early as 3 days after AI, although more accurate results are
obtained if samples are taken later at 7-8 days.

v). Pregnancy associated glycoproteins (PAG)

Pregnancy specific proteins are known to be produced in various ruminant species

including cattle, buffalo, sheep and goats. Two pregnancy specific proteins (PSP) A and B have
been isolated from bovine fetal membrane extracts by Butler et al. (1982). Of these PSP-A was
identified as a fetoprotein and PSP-B was found to be specific to the placenta. These molecules
appear in the maternal circulation and can be determined with accuracy from 24 days of gestation
onwards. The PAG continues to be existent in maternal blood for the entire pregnancy and up to
100 days post partum. The assay involves radioimmunoassay on serum. The limitations to the
wide spread use of this test is non availability of the protein in urine, presence of PAG up to 100
days (i.e false positive) and the non availability of cow side commercially available kits for its
detection. Recently, the existence of PAG has been documented in bovine milk.

C). BIOLOGIC TESTS

i). For eCG: Several biologic tests were developed for the detection of eCG including the
Aschiem Zondek Test, the Friedman test (rabbit test) or the Frog or toad test.

Aschiem Zondek test: Aschheim and Zondek described a test (known as the A-Z test)
which identified the presence of eCG in serum of pregnant mare wherein serum from mares is
injected to mice and the results are read later. The serum (0.5 ml SC daily for 2 to 4 days or 5 ml
intraperitoneally) from test mare is injected to 2 to 3 rats (22 days of age) and rats are killed (72 hr
when injected intraperitoneally and 96 to 120 h later when injected subcutaneously) and a positive
test is indicated by the presence of multiple corpora haemorrhagica on the ovaries and uterine
edema. The test was considered to be 90 percent accurate when performed between the 60 to 100
days of pregnancy in the mare.

Friedman Rabbit test

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Serum from test mare is injected (2 ml given IV) to rabbits (14 to 20 weeks age) kept in
isolation and laparotomy performed 24 h later. A positive test is indicated by the presence of
corpus haemorrhagicum and uterine edema.

Toad test

The basis of this test is the concept that the sperm cells are emitted by toads/frogs only
when stimulated with female frogs or gonadotrophins. Two male toads are taken and their cloaca
is wiped with normal saline and examined for presence of spermatozoa. If no sperms are present
the cloaca is cleaned and 1 ml of test serum from a mare is injected into the dorsal lymph sac of 2
male toads thrice at an interval of 1 hour. The cloaca is examined for the presence of the sperms.
A positive test is indicated by the presence of sperms in the cloaca within 1 to 6 hours after the last
injection.

PREGNANCY DIAGNOSIS IN BITCHES

More reliable methods include abdominal palpation (at 30 days gestation), measuring the
level of relaxin in the blood (after 26 days gestation), ultrasound of the abdomen (starting at 21-30
days gestation), and radiographs (after 45 days gestation). These methods vary in their ability to
positively identify the number of fetuses and fetal viability.

1. Relaxin assay

Relaxin can be determined in the peripheral circulation of pregnant bitches at 20-30 days
of gestation, whereas it is absent in non-pregnant bitches at all stages of the reproductive cycle.
This relaxin is known to be produced by the placenta in the bitch and cat and is thought to
contribute to its maintenance by inhibiting uterine activity. In the domestic dog it has been
established as a pregnancy-specific hormone. The site of synthesis in the bitch is primarily
ascribed to the placenta, although the hormone can also be traced in the ovary and uterus.

Note:
 Progesterone concentration in the peripheral circulation of pregnant bitches is similar to
non –pregnant, therefore unlike other species progesterone can‟t be used to diagnose
pregnancy.

2. Ultrasound

Ultrasound is the best method used to visualize and evaluate the pups in the womb.
Ultrasound is best done 21-30 days after breeding. Ultrasound done prior to 21 days can give false
negatives. Ultrasound is a good method of distinguishing between pregnancy and pyometra. Fetal
viability can be determined by ultrasound through visualization of the fetal heart. The number of
fetuses can be estimated, though it can be less accurate in bitches carrying larger litters, or when
done later in gestation.

3. Radiographs
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Twenty-eight days into pregnancy, the bones of the fetus begin to become calcified. At
42-45 days of pregnancy, radiographs can be taken to visualize the pups in-utero. However, for
best results, radiographs should be taken more than 47 days post breeding, when fetal skeletons
are more readily visible. Radiographs can help determine the number of fetuses, though it is less
accurate in bitches carrying large litters. Fetal viability cannot be determined by radiograph unless
advanced lesions exist in the fetus, such as skeletal collapse or gas within the fetus.

In bitches the fetal skull is visible by day 45 and the entire fetal skeleton is visible by the
end of seventh week of gestation. The number and position of the fetuses can be detected easily by
radiography at this time. Signs of fetal death as seen by radiography include the spalding sign,
(which is the overlapping of the cranial bones), gas shadows in the fetal heart and stomach and
tightly flexed spine (seen in fetuses died for long time).

4. Abdominal palpation

Abdominal palpation of pregnancy in bitches may be possible in small or medium sized

bitches which are not too obese. Moreover, bitches often tense their abdominal wall and respire
too fast making abdominal palpation often too difficult to perform. Beyond day 55 it is easy to
diagnose pregnancy by the palpation of the fetuses. They may be palpable in the flank and also in
the lower abdomen.

TERMINATION OF PREGNANCY
Mare:
For the management of twinning, if the pregnancy needs to be curtailed for this and other
reasons like mismating, the treatment of choice is PGF2α or an analogue administered after CL has
become responsive to PG. i.e after days 4 post ovulation and before formation of endometrial cups
i.e before 35 days. Hence it is preferred 10-15 days after mating. Alternatively, intra-uterine
infusion of 250-500 ml normal saline solution during the same period as mentioned above is also
effective because of physical effect in flushing out the conceptus. It also stimulates the release of
endogenous PG.

Cattle
Pregnancy can be terminated from day 4-5 to 150 after ovulation with PGF2α or its
analogue. After day 150 placenta also provides additional progesterone for maintenance of
pregnancy until day 270 of gestation. This implies that in such case PGF2α or its analogue alone
will not be effective. So, long acting corticosteroid either alone or in combination with PGF2α is
required.

Ewe
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Between day 4 and 12, PGF2α or its analogue will be most effective. After day 45-55,
source of progesterone is ovary and placenta. Therefore, during 3rd and 4th month of gestation
induction of abortion is difficult. However, use of progesterone receptor antagonist aglepristone
may be effective. During final weeks of gestation, corticosteroid will be necessary to terminate the
pregnancy.

Sow
Pregnancy can be terminated at any stage with PGF2α , however sometimes repeated doses
may be required.

TERMINATION OF PREGNANCY IN BITCHES

A. OESTROGENS

i). Oestradiol cypionate: It was an oestrogen compound commonly used by veterinarians for the
management of unwanted mating. The treatment protocol involved is administration of 0.25 to
1 mg total dose within 3 days of mating. A dose rate of 0.04 mg /kg is 100 percent successful in
preventing pregnancy in bitches. The total dose should not exceed 1mg.

ii). Estradiol benzoate Pregnancy can be prevented in bitch by strategic use of estrogen during
first 5-7 days after mating i.e estradiol benzoate @ 10µg/kg body weight on day 3 rd, 5th and 7th
sub-cutaneous or i.m.

iii). Diethyl Stilbesterol (DES) (A non-steroidal synthetic estrogen): Injection of DES at a

dose rate of 2mg/kg body weight, up to 25mg once or twice within 5 days of mating is found to be
highly effective in terminating pregnancy.

iv). Conjugated oestrogen: Conjugated oestrogens can be used for the treatment of misalliance
in dogs within 5 days following breeding. They are mainly isolated from mare's urine and hence it
can be called as conjugated equine oestrogen. Commonly available preparation is PREMARIN
0.625mg and 1.25mg tablets. Dosage 1.875mg daily for 3 days.

However, estrogens can result in the development of pyometra by causing cervical relaxation thus
allowing the vaginal bacteria to enter the uterus. Estrogens may also produce dose related bone
marrow suppression. Diethyl stilbesterol, oestradiol benzoate and estradiol cypionate have been
widely used to prevent implantation. But oestrogen preparations are limitedly recommended in
veterinary practice because of their harmful side effects. Oestrogen is thought to predispose
pyometra in bitches by sensitizing progesterone receptors and enhancing binding of progesterone
to the endometrium. It has been also reported that excess serum estrogens can lead to oestrogen-
induced bone marrow destruction.
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B. NATURAL PROSTAGLANDIN: Prostaglandins are found to be highly effective for termination

of pregnancy in bitches after 30 days of gestation. Its use prior to 30 day is not recommended.
Most preferred treatment protocol is 0.1 mg/kg s/c every 8 hours for 2 days and then 0.2 mg/kg
given s/c every 8 hours until abortion complete (5-7 days). This protocol results in the least side
effects. After administration of drug, the dog should be observed for 30 minutes for any side
effects. The dog should be fed 1-2 hours after prostaglandin injection so as to avoid vomiting.
Most of the animals treated will exhibit some of the following side effects like panting, respiratory
distress, excess salivation, vomiting, defecation, stranguria and urination. Normally these side
effects start within 30 seconds to 3 minutes and usually persist for 5-20 minutes. Side effects are
usually severe during the first few injections and side effects will be diminishing after each
subsequent injection. Preparation: Lutalyse, 5ml &10ml vials are available. Concentration:
5mg/ml.

C. PROSTAGLANDIN ANALOGUES: It has greater luteolytic effectiveness at relatively low

doses and decreases the occurrence of myometrial contractions and severity of side effects
compared to natural prostaglandins. It has much greater affinity for the prostaglandin receptors
and has a longer half life than natural prostaglandin. It can be given at the dose rate of 2.5µg/Kg
body weight subcutaneously every 48 hours for 3 to 4 times. In order to reduce the side effects
atropine sulphate can be given at the rate of 0.04 mg/kg body weight subcutaneously 10-15
minutes prior to the administration of drug. Preparations: Inj. Clostenol 2ml, Inj. Pragma 2ml,
Inj. Cyclix 2ml. Concentration: 250µg/ml.

D. DOPAMINE AGONISTS: Dopaminergic agonists are ergotine derivative alkaloid

compounds that exert an antiprolactinergic effect. The ability of dopamine agonists to inhibit
prolactin secretion makes them optimal for milk suppression, either during overt
pseudopregnancy episodes or in the post-partum period. It is well known that prolactin is a
required luteotropic hormone during the second half of canine luteal phase. Therefore, anti-
prolactins can also be used to suppress luteal function in progesterone dependent conditions
such as pyometra, unwanted pregnancy and mammary tumors. Two of the most widely used
dopamine agonists in dogs are bromocreptine and cabergoline, which have a direct action on
D2-dopamine receptors of the lactotrophic cells of the anterior pituitary gland.

i. Bromocriptine: It has been reported to be an effective abortifacient after 35 days of

gestation but not prior to day 30. Two different treatment protocols have been adopted. In
one protocol 0.1mg/kg body weight daily or BID for 6 consecutive days beginning on day
30 of gestation. In the second protocol, 0.03 mg/kg twice daily for 4 days beginning after
day 30 of gestation. Bromocriptine commonly causes inappetence, anorexia, vomiting and
depression. Also the drug is not 100% effective in terminating pregnancy. Emesis is
presumably due to interaction with dopaminergic elements in the emesis centre of the brain
and the ability of the drug to cross the blood brain barrier. The emetic effect may reduce
the absorption of the total dose administered, and thus compromise efficacy. Because of
above reasons the drug has not been used extensively. Preparation: Encript 2.5 mg tab,
Proctinal 1.25 & 2.5 mg tabs.
 P a g e | 72

ii. Cabergoline: It is a long acting dopamine receptor agonist and prolactin inhibitor. It is
highly effective orally and parentrally. A dose of 5µg/kg once daily cause a sharp decline
in serum prolactin concentration and result in abortion without much side effects. The side
effects of cabergoline are milder (compared to those of bromocreptine) presumably due to
the fact that it appears to be a more specific D-2 dopamine receptor agonist and is less able
to cross the blood-brain barrier and have CNS effects. Preparation: Cabgolin Tab 0.25 &
0.5 mg, Camforte Tab 0.5 & 1mg.

E. COMBINATION OF PROSTAGLANDIN AND DOPAMINE AGONIST: Simultaneous

administration of prostaglandin and dopamine agonist is found to be highly effective. Therapy
begins 28 days after first breeding. Cloprostenol is administered subcutaneously on alternative
days at a dose rate of 1 µg/kg and the drug has to be administered three times. Oral
cabergoline has to be given at a dose rate of 5µg/kg body weight for 9 days. This regimen
reduces the adverse effects of prostaglandin therapy alone and increases the efficacy of
prolactin antagonists.

F. DEXAMETHASONE: Dexamethasone administration beginning at mid-gestation can

terminate pregnancy in dogs, presumably by activating endogenous mechanisms similar to
those involved in parturition. It can result in the production of oestrogen and prostaglandin by
the fetoplacental unit. Single dose of glucocorticoids are not efficacious in the bitch. It can be
given at a dose rate of 200µg/kg body weight for 7 days and then at a tapering dose of 10-20
µg/kg for next 3 days. Advantages of such a therapy for pregnancy termination include the
fact that it involves only oral administration of a relatively inexpensive drug; however
repeated administration and high doses make the method impractical. Preparation: Dexona
Tab 0.5mg, Wymesone Tab 0.5 mg.

HORMONES OF REPRODUCTION

Hormone is a physiologic, organic, chemical substance synthesized and secreted by

specialized cells or glands and is transported through the blood at the target site where itexerts the
action. These are secreted by ductless endocrine glands. Hormone binds at receptor which is
specific.Hormone can be:

a). Primary hormones: These directly regulate various reproductive processes like oogenesis,
ovulation, fertilization, implantation, gestation and parturition.

b). Secondary hormones:These control the physiological function of other system which may
directly or indirectly influence the reproductive process /system. These hormones are also called
as metabolic hormones.

Based on the structure the hormones can be-

1. Protein hormones: These are polypeptide hormones ranging from a molecular weight of
300 up to 70000 daltons. These are soluble in water and these are denatured easily by
heating or by acid or by alkali. These are not active if given or administered orally. e.g.
oxytocin
 P a g e | 73

2. Steroid hormones:Cholesterol is the precursor of these hormones and have a molecular

weight of about 300-400 daltons e.g. testosterone. These are soluble in oil not in water.
These may be active if given orally.
3. Fatty acids:These are derived from Arachidonic acid and have a molecular weight of
about 400 dalton.
4. Amines: These compounds are derived from tyrosine or tryptophan e.g. melatonin.

Hypothalamus occupies a very small portion of the brain. It consists of the region of the
third ventricle.Pituitary is present below the hypothalamus in the depression of sphenoid bone
known as sella turcica. There are neural connections between the hypothalamus and the posterior
lobe through the hypothalamic-hypophyseal tract and vascular connections between the
hypothalamus and the anterior pituitary lobe. This system transports the hormones from the
hypothalamus to pituitary.

The pituitary has two lobes: Anterior lobe or adenohypophysis and posterior lobe or
neurohypophysis

The adenohypophysis has two types of cells:

a) Chromophores: These are round and clear cells and they don‟t take any stain.
b) Chromophils: These can be stained and these can be basophiles and acidophiles. These are
stained by acidic stain(eosin).

Acidophiles can be orangeophiles- from these cells growth hormone is synthesized.

Carminophiles–from these cells prolactin (also known as luteotropic hormone or luteotropinor
LTH) is synthesized.

Basophiles- These are stained by basic dye (Haematoxylin). These can be-

a) Gonadotrophs- These produce FSH and LH.

b) Thyrotropins- These produce TSH.
c) Corticotrophs- From these cells ACTH is produced.

In the blood the pattern of hormones may be:

Pulsatile- Concentration of hormones changes several times within an hour. e.g. LH &
glucocorticoids.

Circadian- The concentration of hormones remain constant for atleast 24 hrs e.g. cortisol
hormones in mare.

Based upon the concentration and time interval the level of hormones may be –

Basal level- In this low concentration of hormone remains constant for a longer period.

Pulse- A slight high concentration remains constant for less than one hour.

Surge- In this a very high concentration remain constant for more than one hour. e.g.
preovulatory surge of LH during estrus period. It remains high for 6- 7 hours.
 P a g e | 74

 For protein hormones the receptorsare present at surface of cells and for steroid
hormones receptors are present at nucleus.

There are four modes of intercellular communications:

1. Neural communication: In this neurotransmitters are released at synaptic junctions from

nerve cells and act across narrow synaptic clefts between as neurotransmitters.
2. Endocrine: In this, hormones are transmitted through blood circulation, typical of most
hormones.
3. Paracrine communication: In this, the products of cell diffuse through extracellular fluid
to affect the neighbouring cells that are at a distance. e.g. prostaglandins.
4. Autocrine communication: Cells secrete chemical messengers that bind to receptors on
the same cell that secreted the messenger.

Neuro-endocrine reflex: Nervous system also controls release of certain hormones through
neural pathways. e.g. Oxytocin in milk let down and LH release following copulation in induced
ovulators.

Principles of hormone therapy:

Hormones are used for therapeutic as well as growth promoters. The action of hormone
depends upon its half life and it varies with species and individual. The major problem of
hormonal therapy is residual effect. The residual effect depends upon the metabolic rate. The
maximum concentration of hormone is always present in liver and kidney. The steroid hormone
remains for a longer period in liver & kidney as compared to protein hormones.The action of
hormone is different in different species. For example hCG & natural LH are effective in all
species whereas PMSG (eCG) is effective in all species except mare so, not used in mare.The
concentration of hormone in the blood also depends upon the structure of hormone; if oily
preparation of progesterone is given intramuscular its level in the blood remains high for about 5-6
days. The action also depends upon the dose of hormone for e.g if you give low doses of estrogen
then it causes contraction of uterus and helps in elimination of infection but if given in high doses
it causes regression of CL so, can be used for the treatment of uterine pathology like pyometra,
mummification of foetus.

Hypothalamic hormones

1. GnRH/ FSH-RH / LH-RH: Schally and Guilemin (1977), two American scientists shared
the Noble Prize for their independent research on determining the chemical structure of
GnRH. It is a decapeptide (10 amino acids) having a molecular weight of 1183 dalton. It is
synthesized and stored in the medial basal hypothalamus. The half life is 7 minutes. It
controls the function of anterior pituitary.
Main function:It causes release of FSH and LH.
Clinical applications / Therapeutic Uses:
i. It can be used to induce estrus.
ii. For treatment of delayed ovulation or anovulation
iii. For treatment of follicular cyst
iv. Treatment of low libido in male animals.
v. Used for treatment of impaired spermatogenesis
 P a g e | 75

Preparations- Receptal, Fertagyl, Ovulanta, Gynarich.

Dose:10- 20 ug.

2. Oxytocin- it is nonapeptide (9 amino acids). Half life of 3-5 minutes, it is synthesized and
produced in hypothalamus by SON(supraoptic nucleus)& PVN (paraventricular nucleus)
but it is stored and released by posterior pituitary i.e it is also called as posterior pituitary
hormone. It is also secreted by corpus luteum. Thus, oxytocin has two sites of origin, the
ovary and the hypothalamus.

Main Functions:

i. Causes contraction of smooth muscles of uterus

ii. Contraction of oviduct thus helps in gamete transport.
iii. Contraction of myoepithelial layer of the alveoli thus displaces the milk from alveoli
into the duct & result in milk letdown.
iv. The stretching of the cervix at parturition caused by the passage of fetus stimulates a
reflex release of oxytocin (Ferguson‟s reflex).
v. Ovarian oxytocin is involved in luteolysis process.

Clinical applications:

i. Used for milk let down

ii. For treatment of uterine inertia.
iii. For treatment of retention of placenta.
iv. In mare used to induce parturition.

Preparations: Gynotocin, Pitocin, Biotocin, Syntocin

Dose: 10-15 IU, i/m. High dose will cause spasm of uterus. Repeat it after every 3-4 hrs
because half life is very short.It should be used very slow i/v but dose is 40-60 I.U in 500 ml
NSS for a period of 3-4 hrs.

Gonadotrophic hormone

From anterior pituitary 3 main gonadotrophic hormones i.e FSH, LH &Prolactin are released.
Their release is controlled by steroid and inhibin hormones.

1. FSH- It is a glycoprotein hormone. It has two polypeptides chain known as alpha & beta
subunits. Presence of both the chains is essential for the action of FSH. The half life 2-2.5
hours.

Main Functions:

i. Causes growth of follicles.

ii. Growth of seminiferous tubules.
iii. Required for spermatogenesis up to secondary spermatocyte stage.
iv. In human being after menopause FSH is excreted in the urine called hMG (Human
menopausal gonadotropin), also used as fertility hormone in domestic animals.

Clinical applications:
 P a g e | 76

i. Used to induce estrus.

ii. For superovulation.
iii. Treatment of impaired spermatogenesis.

Preparations: FSH-ovine (P), FSH – porcine (P), FSH-equine (E).

Dose: 30-40 mg (Total Dose) and given in divided dose for a period of 4-5 days.

2. LH: Also called as ICSH(Interstitial cell stimulating hormone) in case of male. It is a

glycoprotein hormone & has two subunits alpha and beta. Half life is 30 minutes & the
molecular weight is 30 k Dalton.

Main Functions:
i. Causes maturation & ovulation of the follicle.
ii. Luteotrophic hormone (helps in formation and maintenance of CL).
iii. Stimulate Leydig‟s cells which produce androgens and maintain libido.

Clinical application:
i. Used for treatment of follicular cyst.
ii. For treatment of delayed ovulation.
iii. For treatment of low libido in male animal.

Preparation: LH-O, LH-P, LH-E.

Dose: 30-40 mg in divided doses for 4-5 days.

3. Prolactin: Similar to Growth hormones and Placental lactogen. It is a polypeptide hormone.It

helps in lactogenesis. It acts on the CNS of female and induces maternal behaviour. Half life is
15 minutes.Prolactin initiates and maintains lactation. It is regarded as a gonadotropic hormone
because of its luteotropic properties (maintenance of corpus luteum) in rodents. However, in
domestic animals, LH is the main luteotropic hormone, with prolactin being of less importance
in the luteotropic complex.

PLACENTAL HORMONES

From the placenta many gonadotrophic hormones are produced. The structure and function
are similar to gonadotrophins which are produced by anterior pituitary.

1. hCG(Human chorionic gonadotropin): Produced by the chorionic membrane of placenta &

excreted in urine of pregnant women. It can be detected in urine as early as 8 days after conception
& in women it is used as a hormone for pregnancy diagnosis. It is found in both blood and urine. It
is a glycoprotein & has two subunits alpha & beta. The main functions are similar to LH but also
have some FSH activity.

i. It causes maturation of follicle, ovulation & CL formation.

ii. In male, cause secretion of testosterone.

Clinical applications:

i. Used as reliable assay for Pregnancy diagnosis in women.

ii. For treatment of follicular cyst.
 P a g e | 77

iii. Treatment of delayed ovulation.

iv. Treatment of low libido in male animals.
v. Detection of remnant of testicular tissues in castrated dog & cat.

Preparations: Chorulon- 1500 I.U. in delayed/anovulation and 3000 I.U. in follicular cyst

3. PMSG: Also called as eCG (equine chorionic gonadotropin). It is present in blood of

pregnant mare broadly between days 40-140 of gestation. It is produced by endometrial
cups. Usually, there are about 12 cups present at the junction of gravid horn and body as a
circumferential band. These are formed by attachment of trophoblast of chorion with
endometrium of uterus. PMSG is first detected first in the blood on day 38-42 after
ovulation, reaches maximum at 60-65 days declines thereafter and disappear by 150 days
of gestation. It circulates in the blood of the pregnant mares and not excreted in the urine.
It has both FSH and LH biologic actions with FSH actions being dominant. The PMSG is a
glycoprotein & it has alpha & beta subunits. The half life is about 7 days. It is a
glycoprotein with alpha, beta sub-units similar to FSH and LH but with a higher
carbohydrate (especially, sialic acid) content. The higher sialic acid is responsible for long
half life of eCG. In pregnant mare it causes growth of follicles which either ovulate or
become luteinized (without ovulation CL is formed) & thus it forms secondary CL or
accessory CL. These accessory CL produce progesterone which helps in maintenance of
pregnancy in mare. The functions are similar to that of FSH. eCG was one of the
commercially available gonadotropins used to induce superovulation in farm animals.
Preparation: Folligon 1000 I.U.

 In mare, the main source of progesterone, in early pregnancy is the true CL/CL verum
(formed from the follicle that ovulated and generated the oocyte that was fertilized) and the
accessory CL. The true CL is active for first three months of gestation and regress at the
same time as accessory CL. The placenta takes over the production of progesterone after
regression of accessory CL.
 The conceptual bulging in first month of gestation in mareis ventral whereas dorsal in case
of cattle and buffalo.
 Bilateral ovariectomy in 55 days pregnant ewes will not result in abortion because by this
stage of gestation, the placenta takes over the major role of progesterone production.

4. Placental Lactogen:Similar to growth hormone and prolactin. It has lactogenic activity &
prolactin like action. It is glycoprotein & has alpha and beta subunits. It regulates the nutrition
of foetus from the dam.It also regulates the growth of mammary gland. It stimulates
progesterone synthesis.PL is detected in the serum of pregnant animal in the last trimester of
pregnancy. It is more important for its growth hormone properties than its prolactin properties.
It is important in regulating maternal nutrients to the fetus and possibly it is important for fetal
growth.

5. PSP(Pregnancy specific protein) also known as PSP-bovine (PSP-B) or PSP ovine.It can be
extracted from placenta during early gestation. In bovine it appears from day 24 of gestation &
the level remains constant throughout the pregnancy. It is used as an assay for early pregnancy
 P a g e | 78

diagnosis. This placental hormone has the potential to be the first reliable hormonal pregnancy
test for cattle.

GONADAL HORMONES

1. Relaxin: it is a polypeptide & also has alpha and beta subunits connected by two disulfide
bond. It is similar to insulin but the biological properties are entirely different. It is mainly
produced by CL of pregnancy. In smaller amounts it may be produced by the uterus & placenta.

Main functions:

a. Causes relaxation of uterus, cervix & vagina during parturition.

b. Cause relaxation of pubic symphysis and dilatation of cervix.
c. Causes growth of mammary gland if given in conjunction with estradiol.
d. Inhibit uterine contraction.

2. Inhibin: also called as folliculostatin. It is a polypeptidewith alpha & beta subunits. It is

produced by granulosa cells of follicle & sertoli cells of testis. Due to negative feedback on the
hypothalamus & pituitary it controls the release of FSH. In male, inhibins are released via the
lymph and not by venous blood as in the female.

3. Steroids: These are mainly produced by ovaries &testis. And, may also be produced by adrenal
cortex & placenta. The precursor for steroid is cholesterol. All the steroids have a common
structure called as cyclo-pentano-perhydro-phenanthrene ring.
a) Androgens: are 19 carbon steroids. Most common is testosterone. It is produced by
Leydig‟s cells of testis & also produced by adrenal cortex. In equine it is produced in large
amount by epididymis so, if during castration a small tissue of epididymis is left the
castrated stallion will behave like a normal stallion, because androgens are produced by the
remaining epididymis. Allowing part of the epididymis to remain is termed “cutting a
horse proud”. In blood the testosterone is bound with globulin & it is also called as SBG
(steroid binding globulin). About 97-99% of the testosterone is bound with globulin and
only 1-3 % is free & this free testosterone is responsible for biological action. It enters the
cells and is converted into DHT (Dihydrotestosterone). The enzyme involved in this
conversion is 5α-reductase. DHT is responsible for the action of testosterone.

Main functions:

i. Causes growth of male genitalia.

ii. Maintain the secondary sexual characters.
iii. Acts as a growth promoter (Anabolic hormone).
iv. Maintain the libido.
v. Required for spermatogenesis after the secondary spermatocyte stage.
vi. Prolongs the life of spermatozoa in epididymis.
vii. Cause growth of accessory sex glands.

Clinical applications:

i. Used for treatment of impaired spermatogenesis.

ii. Treatment of low libido in male animals.
 P a g e | 79

iii. To produce cow steer (castrated male) which are used for heat detection.

Preparations: Testobion

b). Estrogens:It is an 18 carbon steroid hormone. These are mainly produced by the ovary & in
small amount may also be produced by placenta & adrenal cortex. For the synthesis of
estrogen there are two cells two gonadotrophin hypothesis or theory-

Male LH Female

(Testicular tissue ) Cholesterol (Ovarian tissue)

Leydig‟s cells Testosterone

FSH

Sertoli cells Testosterone Granulosa cells

Estrogen

Aromatization: Conversion of testosterone into estrogen.

Main functions:

1. Causes growth of uterus.

2. Helps in maintaining secondary sexual characteristics.
3. Used as growth promoter (anabolic hormone).
4. Causes the growth of ducts of mammary gland.
5. Act on CNS to induce behavioural estrus in the female, however, small amount of
progesterone along with estrogen is also needed.
6. Act on uterus to increase both amplitude and frequency of contractions by potentiating the
effects of oxytocin and PGF2α.
7. Luteolytic in cattle, buffalo and sheep but luteotrophic in sow.
8. It helps in calcium uptake.
9. It causes maturation of epiphysial cartilage of long bones due to which after attaining
puberty there is no increase in height of female.

Also produced by some plants mainly the legumes (berseem) & these are known as
phytoestrogens. If the berseem is fed in excess it may cause infertility. Diethylstilbestrol (DES), a
synthetic non-steroidal estrogen formerly used as growth promoter in cattle and sheep binds with
estrogen receptor and acts with same potency as 17-beta estradiol. Because of its carcinogenic
effect, it has been replaced with other estrogenic implants.

Clinical applications:
 P a g e | 80

1. Used to induce estrus.

2. Used to induce abortion.
3. Used for treatment of endometritis & ROP
4. High dose use for treatment of pyometra & mummification of foetus.
5. In bitch – treatment of mis-mating
6. Used as oral contraceptive.

Preparations: Progynon depot – 10mg/ml have estradiol valerate.

Mixogen- contain estradiol benzoate, preparation are 4, 20 & 40 mg.

c). Progesterone: It is a 21 carbon steroid hormone secreted by CL & also by placenta & adrenal
cortex. It is the most prevalent, naturally occurring Progestogen.

Functions:

i. Growth of alveoli of mammary gland.

ii. Regulate estrus cycle.
iii. Prevent contraction of uterus.
iv. Prepare the uterus for implantation and maintenance of pregnancy.
v. Acts synergistically with estrogens to induce estrus behaviour.
vi. Inhibits estrus and ovulatory surge of LH at high levels.
vii. Main hormone for regulation of the estrous cycle.

Clinical applications:

i. Used to induce & synchronize estrus.

ii. Used to prevent abortion.
iii. Used as contraceptive pill which prevent the ovulation.

Preparations: Proluton depot, Duraprogen, P-depot, Hyprogen. Preparations are in the 2 and 3 ml
containing 500 mg and 750 mg, respectively.

UTERINE HORMONES

Prostaglandins, first isolated from accessory sex gland fluids, were termed prostaglandins because
of their association with the prostate gland.PGs are 20-carbon unsaturated hydroxyl fatty acids.
Arachidonic acid & linolenic acid are the precursor of PGf2α.PGF2α is natural luteolysinthat causes
regression of CL. Endomethacin is PGF2α antagonist it prevent synthesis of PGF2α, and prolongs
the life span of CL. Most of PG they act locally at the site of production but sometimes they may
be transported to the target organs away from the site of production.

Functions-

1. Causes regression of CL.

2. Helps in ovulation.
3. Contraction of myometrium.
4. Help in gamete transport to site of fertilization.
 P a g e | 81

Clinical applications:

1. Induce abortion.
2. Induce parturition.
3. Treatment of pathological conditions like pyometra in cattle and buffaloes.
4. Treatment of fetal mummification
5. Used in estrus synchronization protocols.

Preparations:

Natural (Dianaprost) e.g. LutalyseR:5 ml (25 mg) i.m

Synthetic (Cloprostenol) e.g. Vetmate, Metrum, Pragma, Repragna - 2ml vial (500µg)

Pheromones: These are also called as sex attractants. These are chemical substances produced by
exocrine glands. These are low molecular weight fatty acid derivatives. These are released in
environment where they are ingested or absorbed or perceived by olfaction and then evoke a
specific response in that animal. These are intra-species (act between same species). These are
not hormones because hormones are produced by endocrine glands and act within the
individual. Pheromones can be:

1. Signalling Pheromones: Their action is rapid and acts through the CNS and their effect is
reversible. For example if two groups of unfamiliar mice ate kept together, they become
aggressive and bite each other. This is because of secretion of pheromone from urine and
foot pad of the mouse. If their nasal epithelium is destroyed, then aggressive behaviour is
lost.

Australian rabbit produces a pheromone from chin gland which is repellent means
prevent the entry of other male in his territory.

2. Priming pheromones: They require a prolonged stimulation for action and their response
is irreversible or permanent. For example –
a. In honey bees, the queen bee produce a pheromone called 9-ketodecanoic acid
which is ingested by other females and prevent the development of the ovaries. So
all the females become sterile and act as workers.
b. Lee-Boot effect: If a group of female mice are kept together, then their estrous
cycle is disturbed and estrus is followed by prolonged anestrous.
c. Whitten effect: If a male is introduced in a group of female mice, their estrous
cycle is shortened and it is called as whitten effect.
 P a g e | 82

d. Bruce effect: If a strange male is introduced in a group of pregnant female mice,

then abortion occurs.

Bartlett technique for sample collection???

Janet’s syringe used for collection of Trichomoniasis suspected samples.

Fertility: Fertility is defined as ability and desire of female to mate, capacity to conceive, nourish
the fetus and finally the power to expel the live fetus. Fertility is expressed by producing a live
calf in a year.

Sterility: It is permanent inability to reproduce or permanent loss of fertility.

Infertility: Infertility is reduced fertility which results in delay in producing a live calf in a year. It
is also defined as temporary loss of fertility.

INFERTILITY can be

I. Anatomical/ Congenital/ Genetic

II. Infectious
III. Functional
IV. Managemental

I. ANATOMICAL/ CONGENITAL/ GENETIC:

a). Ovarian hypoplasia: It is due to recessive gene. It can be unilateral or bilateral, complete or
partial. In majority of cases left ovary is affected more than right ovary. In affected ovary, the
number of primary follicles is reduced. In unilateral hypoplasia the animal is normal cyclic and
genitalia is normal. And the affected ovary is smaller in size. In bilateral hypoplasia, genitalia is
poorly developed and animal is anestrus. Affected ovaries are pea sized and difficult to be palpate.
There is no treatment of this condition. Animal affected with unilateral ovary should not be used
for breeding because of its transmission to next generation.

b). Ovarian agenesis: Also called as gonadless condition. Both the ovaries fail to develop and
genitalia is poorly developed. Condition is diagnosed at the time of puberty when animal fails to
exhibit estrus. There is no treatment for this condition.

c). Congenital lack of endometrial glands: It is mostly observed in cross-bred heifers. Animal
exhibit estrus once and then becomes anestrous because the endometrial glands are absent which
are responsible for PGF2αsynthesis which causes regression of CL. In such cases if PGF2αis
administered animal exhibit estrus but again become anestrus. If such animals are inseminated,
they may conceive but after sometime abortion occurs because the caruncular areas are also absent
in such cases and due to this proper placentation does not occur.

d). Segmental aplasia of mullerian/paramesonephric duct:It is an inherited,congenital defect

that arises due to segmental aplasia of mullerian duct leading to a wide range of vaginal,cervical
 P a g e | 83

and uterine defects.The incidence of such conditions is higher in exotic than Indian cattle and
buffalo. The aplasia may be in any breed and anywhere along the duct system;however the
cervical area is more affected.The remainder part of uterine horn often gets filled up with
secretion.The ovaries develop cyclic behaviour with normal secretary activity and level of
steroids.Sometimes only one uterine horn has a lumen, the other appearing as narrow,flat band,the
condition is referred as uterine unicornis.It is more common for the right horn to be absent than
the left. Animal may conceive provided ovulation occurs from non- effected side of uterine horn.A
more serious type of aplasia occurs when isolated secretions of uterine horn are present. Uterine
secretions accumulate and cause sac like dilation of such isolated portion of the tract.Animal with
this condition are sterile. This can become very large and can be confused with early pregnancy
during per rectal examination. Animals with this deformity are sterile.

Congenital abnormality of cervix may involve duplication of the lumen of cervix,each

uterine horn connects with the vagina by a separate cervical canal. Affected animals conceive in
normal fashion but show dystocia. In heifers single cervix may open into double os uteri externum
with a dorso-ventral post cervical band. In case of uterine didelphys, a double cervix is present;
the uterine body is divided and division extends up to the cranial part of the vagina. Such animals
may conceive provided they have been inseminated in the horn ipsilateral to the ovulation. The
most common developmental aberration of the female tubular organs/ mullerian duct system
involves a variable degree of persistent hymen, appearing as a vaginal constriction in front of the
urethral opening.

White heifer disease was so named because it occurs most commonly in white heifers of
the shorthorn breed. Hence, segmental aplasia leading to Persistent hymen or imperforate hymen
is also called as white heifer disease because of the association of this genetic defect with white
coat colour Condition is caused by single, recessive, sex limited gene with linkage to the gene for
white colour. In case of complete hymen obstruction, there is accumulation of secretions causing
fluctuating swelling of variable size that can be palpated per-rectally. Animal may develop fever
and straining if secretions are infected with pyogenic organisms. The condition is diagnosed by
per-rectal and ultrasonographic examination. Because of hereditary origin such animals should be
culled.

Parovarian cysts are remnants of the mesonephric ducts that are sometimes present in
the mesosalpinx of cows.

e).Abnormal Wolffian duct: It is called as Gartner‟s duct. These are remnant of wolffian duct.
These ducts are two in number and are present under the mucosa of floor of the vagina. These
ducts are harmless and do not interfere with mating or insemination or parturition but some time
due to infection these become larger and cystic, called as Cul-de-sac. These can interfere with
insemination or mating. These can be removed easily by surgery.

f). Free-martinism: If there are dizygotic twins and one is male and second is female then, the
female is sterile, known as Free Martin. Free-martinism is a distinct form of intersexuality that
arises as a result of vascular anastomosis of the adjacent chorioallantoic sacs of heterozygous
fetus. The vascular anastomosis occurs as early as 30 days of gestation. Due to anastomosis fetal
androgens from male are diffused to female side which suppresses the development of female
genitalia. If there is death of male twin of a heterozygous pair after this time with the other being
 P a g e | 84

carried to term. Therefore, it is possible for a single born freemartin to occur. The external
genitalia of freemartin heifer may appear normal; the internal genitalia are grossly abnormal. The
gonads are typically vestigial but in minority of cases have undergone masculinization and the
gonads resemble testes (parenchyma contains recognizable tubules and interstitial tissue).
Structures developed from paramesonephric duct are absent or hypoplastic. Development of
mesonephric ducts is related to the degree of masculinization of gonads. Vestigial gonads of
freemartins are devoid of follicle and oocytes.

The newly born freemartin can be recognized by prominent clitoris with an obvious tuft of
hairs at the ventral commissure of vulva, although these signs are not always reliable. In adult
female the length of vagina is about 30 cm compared with 8-10 cm in freemartin. In calves of 1-4
weeks of age the vagina is normally 13-15 cm in length compared with 5-6 cm in a free martin. It
is common in cattle than other species.The most accurate method of diagnosis is demonstration of
sex chromosome chimerism in cultured lymphocytes.The condition is generally diagnosed at the
time of puberty when female fails to exhibit to estrus. The free martin is generally a female
because it contains bar bodies. In such cases WBC show 90% XX/XY chimera. Chimera:
Individual having the cell lines of two different animals. On rectal examination, the ovaries,
uterus, cervix and a part of vagina is absent. The animal is steer like (castrated male) and external
genitalia (vulvar lips) is apparently normal. There is tuft of long hairs on vulvar lips. The vagina is
not patent (open) because part of vagina is missing.For free-martinism various theories have been
postulated:

i. Hormonal theory: It was proposed by Lillie. According to this theory, from the
male fetus the androgens produced by testes passes to the female and is responsible
for free martinism in female fetus.
ii. Cellular theory: According to this, the primordial germ cells which migrate from
the yolk sac to the genital ridges may pass to the female fetus and these prevent the
growth of female genital tract.
iii. Chemical theory: According to this, from the medulla part of the gonads,
medullarian substance and from cortex cortexin is produced. Testes develop earlier
than the ovary. So the medullarian produced by the testes passes to the female and
prevent the growth of the genitalia.
iv. Latest theory: There is H-Y antigen in the male fetus. The H-Y antigen passes to
the female fetus and prevent the growth of genitalia. In free martin, the ovaries are
not developed or absent. The uterine horn or body is also absent. The cervix is also
absent. Only a part of vagina is present. It can be diagnosed by-
i. By history of twinning (male and female)
ii. By rectal examination where the genitalia is absent
iii. Fincher test: In this a glass tube or speculum of diameter of 3/8 to ½ inches is
inserted in the vagina and in case of free martin it will not go beyond 7.5-10 cm
whereas in normal animal it may go upto 15-20 cm.
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iv. Demonstration of sex chromosome chimerism in cultured lymphocytes.

v. Diagnosis by PCR for the presence of Y-chromosomes in blood cells.

g). Hermaphrodite/ Inter-sexes: The inter-sexes are animals with ambiguity of sex. These have
abnormal genitalia. These can be true hermaphrodite and pseudo-hermaphrodite. True
hermaphrodite contain gonads of both the sexes (i.e. ovo-testis) and external genitalia may be of
male or female. Pseudo-hermaphrodite can be male or female pseudo-hermaphrodite. Male
pseudo-hermaphrodite has testes; external genitalia of female and behaves like a male. In case of
female pseudo-hermaphrodite animal contains ovaries, external genitalia of male and behaves like
a female.

h). Tumors: Granulosa cell tumors and fibromas are generally the most common neoplasms of the
bovine ovary.Most common is granulosa cell tumor. It may be unilateral or bilateral. It is common
in cattle and mare. During initial stages the animal exhibits nymphomania because of continuous
production of estrogen. After few days lutenization occur and there is production of progesterone
due to which animal remain anestrous.

II. INFECTIOUS INFERTILITY: It can be due to specific and non-specific infections.

A. Non specific infections:Non-specific infection is present in cattle and mostly they are non-
pathogenic and opportunistic organisms. If there is predisposing factor, they may cause infection.
This may lead to infection of genital tract and abortion. The infection mostly affects individual
animals. Mostly the organism in non-specific infection are Pseudomonas, Streptococcus, Staph.,
Salmonella and Pasteurella.

a). Oophoritis: The infection of ovary occur due to

i. Ascending or descending infection .

ii. Rough manipulation of genitalia during rectal examination.
iii. Ovarian massage.
iv. Manual removal of CL.
v. After inflammation of ovary, in most of the cases adhesions occur and the animal
becomes repeat breeder. There is no treatment for this condition. But it can be
prevented by avoiding rough manipulation of genitalia.
vi. Tuberculous oophoritis, brucella induced oophoritis and ovarian abscessation in
animals sometimes leads to generalized pyaemia.

b). Ovario-bursal adhesions: These are adhesions between ovary and oviduct. It may occur due
to

i. Oophoritis.
ii. Ascending or descending infection.
iii. Manual removal of CL.
iv. Excessive mutation at the time of dystocia.
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v. If large quantity of irritant solution is infused in uterus.

Adhesions may be unilateral or bilateral. In unilateral adhesions animal may conceive but
in bilateral cases animals fail to conceive and become repeat breeder. There is no treatment
for these adhesions. To find out the patency or opening of the fallopian tube, tubal patency
test can be conducted as following:
Phenol Sulphonaphthaline (PSP) and Indigocarmine Test: If PSP is induced in uterus,
it is not absorbed and passes through fallopian tube into peritoneal cavity where it is
absorbed and excreted into urine. Using Foley‟s catheter 20 ml of 0.1 % PSP is infused
intra-uterine in one horn preferably at the tip of horn otherwise the dye gets eliminated
through external os. After 30 minutes to 3 hours the urine is collected by catheterization as
the dye is absorbed from peritoneum and excreted with urine and the colour of urine is
recorded. If the fallopian tube is blocked, colour of the urine will not changed. If it is not
blocked PSP will pass into peritoneal cavity and excreted by kidney into urine and urine
will turn pink. This colour will remain pink for 12 hours. If urine is not alkaline, it is made
alkaline by adding 0.2 ml of 10% Tri-sodium orthophosphate to 10 ml urine. Test is
preferred to be carriedout for another horn during next estrous cycle. The test is generally
conducted in luteal phase preferably at about day 10 as false positive can be obtained in
follicular phase. If you want to test both the horns at same time, then you have to
catheterize and remove complete urine from bladder after 6 hours of testing the first horn.
Second horn can be tested by dissolving 1000 mg of Indigocarmine in 100 ml distilled
water and 10 ml of this dye in the horn at the tip and collect urine after 2-3 hours by
catheterization. If urine turns blue means there is no blockage.
Starch test: Take 1g of sterile starchand dissolve in 10 ml of distilled water. Infuse it
around the periphery of ovary with the help of a 18 gauge needle at estrus. After 3-4 days
collect the mucusfrom the vagina. Take this mucus on slide and add 2-3 drops of 2%
lugol‟s solution. If it is positive the colour will become blue. Similarly, perform this test
for other ovary. A blue colour of the sample indicates patency. Bilateral occlusion of
oviducts causes animal to be irreversible sterile.
Tubal blockage can be treated by CO2 insufflation to unblock the oviducts but risk of
rupture of these ducts. Therefore no successful treatment is there.
c). Hydrosalpinx: Accumulation of water in fallopian tube. It mostly occurs due to adhesions of
fallopian tube and in case of segmental aplasia (White heifer disease). If hydrosalpinx is bilateral,
then animal is repeat breeder. On rectal palpation the fallopian tube is enlarged and wide. There is
no treatment for this condition.

d). Pyosalpinx: Accumulation of pus in fallopian tube. It may occur due to infection of fallopian
tube. Second, if case of pyometra is repeatedly treated by large doses of estrogen. Then, pus from
uterus may ascend to fallopian tube. On the basis of per-rectal examination hydrosalpinx and
pyosalpinx can‟t be differentiated.

e).METRITIS:

Uterus has three layers namely endometrium, myometrium and perimetrium and the inflammation
of them is known as endometritis, myometritis and perimetritis respectively. Puerperal Metritis
occurs within few days of parturition and usually follows an abnormal first or second stage of
labour especially when there has been a severe dystocia that has required prolonged traction or
resulted in damage to vulva and or birth canal. The condition is also associated with uterine
inertia, retention of placenta, immature calving (including abortion or induced calving) and twin
births. Endometritis is sequellae of long lasting Puerperal Metritis.
 P a g e | 87

Metritis is the inflammation of uterus involving all the three layers. It occurs mostly due to -

i. After abortion or dystocia or retention of placenta.

ii. If unsterilized instruments are used for AI.
iii. Poor hygienic conditions at the time of parturition.
iv. Poor hygienic conditions at the time of vaginal examination.
v. Due to pneumo-vagina (Accumulation of air in the vagina, common in mare).

It may occur due to ascending or descending infection.

Symptoms: In severe cases pus or mucopurulent discharge come through the vagina. In more
severe cases sometimes animal may exhibit systemic signs like decrease in appetite and milk
yield. On rectal palpation, uterus is enlarged and has thick uterine wall. In mild cases, pus flakes
are seen in vaginal mucus particularly at the time of estrus. Sometimes, there may be sub-clinical
infection. In this the genitalia are apparently normal with normal discharge. It can be only
diagnosed by taking endometrial biopsy where lots of neutrophils are observed. In endometritis
animal is normal cyclic but fail to conceive. Sometimes, in chronic cases animal may become
anestrous which is due to damage of endometrium and failure of synthesis of prostaglandins due
to which CL persists.

Treatment:

i). Cephalosporins have been widely recommended for the intra-uterine treatment of
endometritis. Ceftiofur is one of the latest systemicantibiotics used in treatment of uterine
infections.

 Intra-uterine administration of Nitrofurazone is irritant and has adverse effects on

fertility. Aminoglycosides are not effective in the predominantly anaerobic
environment of the infected uterus. Sulphonamides are ineffective because of
presence of para-amino-benzoic acid metabolites in the lumen of infected uterus.
Penicillins are susceptible to degradation by the large numbers of penicillinase
producing bacteria that are present in the infected uterus. Intra-uterine
administration of tetracyclines may worsensubsequent reproductive performance
due to irritation and pH damage to the endometrium.

Principle for the choice of antimicrobial / antiseptic agents:

 It must be effective against Gram positive and Gram negative bacteria.

 It must be effective within the generally anaerobic environment of the uterus.
 When the intra-uterine route is used, the substance must be evenly and rapidly
distributed throughout the uterine lumen with good penetration into the deeper
layers of the uterus.
 It must not inhibit uterine defense mechanisms particularly the cellular component.
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 It must not traumatize the endometrium. Several of the vehicles used in the
formulation of pharmaceutical preparations can damage the endometrium. e.g.
propylene glycol, which can cause a necrotizing endometritis; oil which can cause
granuloma and chalky bases which can cause irritation and blockage of the glands.
 Treatment must not reduce fertility by producing irreversible changes in the
reproductive system.
 Treatment must be cost effective for enhancing fertility
 Details of its absorption from the uterus and excretion in the milk must be known
so that appropriate withdrawal times can be followed.

ii). Povidone iodine (Betadine):Diluted betadine is used for intra-uterine infusion. Dilution makes
the iodine free. Dilution is also necessary because of its irritant nature. Results are better if
dilution is with metronidazole. Dose of diluted solution is 40-60ml intra-uterine.

iii). Estrogen: 5-10 mg of estrogen is generally recommended and it increases uterine blood
circulation. It increases uterine clearance and elimination of infection.

iv). Prostaglandin:It causes luteolysis, increases uterine contraction and thus helps in expulsion of
pus. Dose of Dianoprost(Lutalyse): 25mg and Cloprostenol(Vetmate): 500µg.

f). Endometritis: It is the inflammation of endometrium, the mucous membrane or internal lining
of the uterus along with its glandular tissue and often occurs as a result of infection by pathogenic
organisms. Infection normally ascends into the uterus via the vagina particularly at service or
around parturition.

Diagnosis:

 Physical examination: Visual inspection may reveal presence of crust of purulent

nature in the perineal region and ventral commisure of the vulva.
 Rectal examination: Most commonly employed method. Accurate diagnosis
through palpation is related to skill, experience and training. The size and
consistency of the uterus and cervix along with presence of fluid in the uterus are
the main criteria.
 Endometrial biopsy: Examination of endometrial changes via uterine biopsy is
another supportive procedure when significant inflammatory changes are evaluated.
Established inflammation produces a superficial lymphocyte and plasma cell
migration.
 Uterine cytology and culture: Detection of significant number of neutrophils is
virtual assurance that inflammation exists. Uterine culture is used to suggest the
etiology of already confirmed inflammation. The alkaline pH of uterine
fluid/sample is indicative of endometritis.
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 Other diagnostic tests: Increased alkaline phosphatase and transaminase in uterine

flushing of animal are indicative of endometritis.
 White side test: It is conventional but reliable method of diagnosis of endometritis.
Test is considered positive when the colour of cervical mucus turns yellow after
boiling with equal volume of 5% NaOH. Intensity of colour depends on the number
of white blood cells present in the uterine discharge.
 Ultrasonography: Real time ultrasonography has been used to demonstrate uterine
changes associated with endometritis. Presence of echogenic particles is indicative
of endometritis. The uterine wall with chronic endometritis is variably thickened.
 Cytobrush: with the help of cytobrush fitted in cytobrush assembly uterine
sample is taken by rolling the cytobrush clockwise and anticlockwise and
smear is prepared, fixed with methanol and stained with Giemsa stain. If PMN
cell count is more than 10% then animal is said to be positive for sub-clinical
endometritis.

Treatment:

i. PGF2α and Intra-uterine Antibiotics

ii. Future therapy with IMMUNOMODULATORS:
E. coli Lipopolysaccharide (E. coli Endotoxin): 100-200µg of E. coli intra-uterine
infusion increases the leucocyte count 100 folds. These are recommended for post AI and
therapeutic purpose in multiple doses in the treatment of sub-clinical endometritis in
bovines. In dystocia cases, instillation of 300 µg after relieving dystocia or caesarean
prevents development of pyometra as well as subsequent endometritis.
Oyster glycogen: 1-10 % oyster glycogen leads to lecocytosis in uterine lumen by 2-30
folds. Intra-uterine infusion of oyster glycogen 500 mg in 50 ml of vehicle leads to marked
improvement in the non-specific uterine defense and endometrial histopathological
pictures in acute and chronic endometritis.
Serum, Plasma and hyper-immune serum: Addition of a small amount serum into uterus
increasesphagocytic ability in uterus. This is by presence of complement, antibodies and
opsonin in the serum. 50-100 ml of autologous plasma for 3 days can be given in the
uterus.
Granulocyte-Macrophage Colony Stimulating factor(GM-CSF): It is a lymphokine
which helps in the differentiation and proliferation of certain hemopoitic cells and
particularly help in formation of mature PMN colonies or macrophage or both. GM-
CSFincreases lyphocytic ability, inhibits their migration and makes them more adherent to
the bacteria.
Inmodulen (LPS+ Propionibacterium granulosum) : Recommended both intra-
muscular and intra-uterine infusion.
Lysozyme: Intra-uterine infusion of 2 mg of lysozyme in 50 mlPBS buffer or 5 % glucose
solution increases INF alpha factor and cause acute phagocytosis. Besides, it also has
antiviral efficacy and modulate TNF alpha synthesis without any mutagenic or allergic
reactions. This can be used for prophylaxis as well as therapeutic purposes. Repeated
infusions are given after 24 to 72 hours if necessary. Available commercial preparation:
Lydium KLP TM (Nika Health Product Ltd.).
 P a g e | 90

g). Pneumo-vagina: It is accumulation of air in the vagina. It can be treated by Caslick‟s

operation. In this, operation upper 2/3 of mucosa of vulvar lips is scarified or removed, then
vulvar lips are sutured by catgut/silk and only 2-3 fingers vulvar lips are left. Sutures are removed
after 7-10 days and there is formation of adhesions. Animal is inseminated artificially and at the
time of parturition adhesions are removed to avoid dystocia.

h). Pyometra: It is characterized by bilateral enlargement of uterine horns due to accumulation of

pus in the presence of active corpus luteum and the animal is anestrus. CL persists due to failure
of endometrium to produce PGF2α.. Condition occurs mostly due to dystocia and retention of
placenta between days 15 to 60 leading to chronic endometritis. The sequel of this chronic
endometritis is pyometra. In some cases pus is seen when animal is lying down. On rectal
examination the uterine wall is thick and contents are also thick. On vaginal examination, the
cervix is closed and small amount of pus may be observed around the external os of cervix. Trans-
rectal ultrasonography will reveal the absence of fetus and the presence of „speckled‟ echotexture
of the uterine contents compared with the anechoic appearance of normal fetal fluids.

Etiology: Brucella , Pasteurella , Pseudomonas, Corynebacterium.Early embryonic death due to

T. fetus results into pyometra. Post coital pyometrais also seen in cases of Trichomoniasis.

Pyometra should be differentiated from normal pregnancy.

Pyometra Pregnancy
Both the uterine Only pregnant horn
horns are enlarged is enlarged
Uterine wall : thick Thin and tonic
and atonic
Uterine contents: Watery
Thick
Fetal membranes Present
and fetus: absent
Fremitus: Absent Present

Treatment: PGF2α. Dianoprost tromethamine (Lutalyse): 5 ml or Cloprostenol (Pragma): 2ml

single injection. After expulsion of pus treat the animal as that of Metritis.

Pyometra in mare: Unlike cows, mares with pyometra do not necessary have a persistent CL and
many may cycle normally. Pyometra may be open cervix or closed cervix. Mare with pyometra
seldom shows overt signs of systemic diseases even when there is 60 litres of exudates in the
uterine lumen.

Persistent mating-induced endometritis in mare (PMIE): is associated with delayed uterine

clearance. At coitus semen along with microorganisms and debris is deposited directly into the
mare‟s uterus. Spermatozoa induce an inflammatory response in the uterine lumen within 24-72
hours so that the environment of the uterine lumen is compatible with the establishment of
pregnancy. It is important not to regard this endometritis as pathological, rather it is physiological
reaction that aids in removing excess sperm and debris from the uterus before the embryo
descends from the uterine horns into the uterine lumen 5.5 days after fertilization. If this
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endometritis persists after 4-5 days of diestrous makes uterine lumen incompatible with
embryonic survival and premature release of PGF2αresulting into luteolysis and decline of
progesterone and an early return to estrus. Resistant mares can eliminate induced infection without
treatment while susceptible mares cannot. Reduced physical drainage may contributeto an
increased susceptibility to uterine infection. In general, reduced endometritis is associated with
advancing age, multiparity; although maiden mares may also be susceptible.Older maiden mares
have an abnormally tight cervix that fails to relax properly during the estrus so that the fluid is
unable to drain and accumulates in the uterine lumen.

Oxytocin is more effective than PGF2αor its analogue in short term for enhancing
uterine clearance. A long acting analogue of oxytocin, carbetocin (Reprocine, Vetoquinol) is
more effective where more prolonged uterine contraction is desired.2 Intramuscular injections of
0.14 mg carbetocin are given at 12-24 hours interval after breeding in mares with marked uterine
edema or free fluid before breeding or in mares with more than 2cm uterine fluid12 hours after
breeding.In cases with relatively large (above 2 cm depth) in uterine lumen or in mares known to
be highly susceptible the saline infusion (1 litre) is a better option and lavage is repeated until the
fluid recovered is clear. Repeated small doses of PGF2αare more effective than a single large dose
in inducing estrus and return to estrus. Non-steroidal Phenylbutazone decrease uterine clearance
by interfering with endogenous prostaglandin release. When sedation is required in a mare where
uterine clearance is a concern, xylazine or detomidine is preferred over acepromazine because of
their enhancement rather than inhibition of uterine motility and clearance unlike acepromazine.

j). Cervicitis: It is inflammation of cervix and occurs due to:

 Injury to cervix during forced traction of fetus or fetotomy.

 Injury to cervix by pipette/ AI gun which is most common.
 May occur due to infection.

Cervix is enlarged on rectal and vaginal examination. Some amount of pus may be
observed at the external os. Animal may conceive at AI but if infection is severe it may spread to
the uterus and cause abortion.

Treatment: Douche the cervix with mild antiseptic solution. Repeat the treatment till animal is
cured.

k). Vaginitis: It may occur due to a) Use of unsterilized speculum or instrument during vaginal
examination. b) Due to injury during vaginal examination or natural mating. c).Following vaginal
prolapse.

Affected animal straining and vaginal examination reveal presence of pus and also injury to the
vaginal examination.

Treatment: Douche the vagina with mild antiseptic solution and also apply any antibiotic
(Soframycin) ointment in vagina. It is better to apply antibiotic ointment along topical local
anaesthetic agent.

l) Mucometra and hydrometra: It is accumulation of mucus and water, respectively in the

uterus. It occurs in the cases of:
 P a g e | 92

 Cystic ovarian degeneration

 Segmental aplasia of mullerian duct
 Persistent hymen.
Uterus is enlarged; uterine wall is thin and atonic. Size of uterine horn may vary
from pregnancy of 2 to 4 months. It may be confused with pregnancy and pyometra. It the
condition is due to cystic ovarian degeneration, it can be treated medicinally and if it is due
to persistent hymen it can be treated with surgically. If the condition is due to segmental
aplasia of mullerian duct, there is no treatment.
m). Parametritis and perimetritis: Inflammation of broad ligaments of uterus is known as
Parametritis whereas inflammation of outer wall (serosal layer) of uterus is known as Perimetritis.
Both the conditions arise due to:

i. Injury to genitalia during rectal examination.

ii. After caesarean section.
iii. Injury to uterus during handling of dystocia cases.
iv. It may also occur in cases of diffused peritonitis
v. Rupture of uterus due to pipette.

Affected animal shows signs of peritonitis. During later stages, adhesions of the oviduct,
uterus and cervix occur with organs of the digestive system. If adhesions are severe animal
becomes sterile. Even if adhesions are mild, animal fails to conceive and there is no treatment of
adhesions.

B. Specific infections:Specific infection is transmitted by AI or natural mating and they are also
known as Venereal diseases. It does not require a predisposing factor. A specific infection may
affect the entire herd or it is enzootic.These include Brucellosis, Campylobacteriosis,
Trichmoniasis, IBR-IPV and epivag infection.

a). Brucellosis: Also known as Bang‟s disease, Malta fever, Crimean fever, Rock fever, Gibraltar
fever and Undulant fever. In cattle and mare the disease is caused by Brucella abortus, inpig - B.
suis, goat - B. meletensis and sheep - B. ovis and dog – B.canis. Most virulent and pathogenic is
B. meletensis.The disease is characterized by abortion during third trimester of gestation.
Organism is very sensitive to disinfectant and high temperature. After abortion organism
disappear from uterus and gets localized in lymph nodes like supramammary, retropharyngeal,
external and internal iliac lymph nodes. Then animal acts as permanent carrier. In male animal the
organism is found in testes, epididymis and seminal vesicles. Major route of transmission is
ingestion of infected material. The principal source of infection is aborting cows in which the
fetus, placenta, fetal fluids and milk are heavily contaminated. In humans disease is known as
undulant fever if raw milk is consumed.

In equines, B. abortusalong with Actinomyces bovis causes poll evil and fistulous
withers. Organism may penetrate through intact skin. Disease is not caused by natural mating
because the vaginal wall is multilayered/stratified at the time of heat, so organism cannot penetrate
and secondly, organism is also non-motile. In AI the semen is deposited directly into the uterus
and if semen is infected with Brucella organism causes infection.
 P a g e | 93

After infection organisms are taken up by phagocytosis and are transported to the
nearest lymph node draining that area. From the lymph nodes by haematogenous route, organisms
are transported to other lymph nodes and if animal is pregnant these are transported to the uterus.
In the uterus the seat of predilection for organism is chorion and fetal fluid because these contain
erythritol which is required for growth of Brucella organisms. Then, it causes placentitis and
subsequently abortion occurs. After few days of abortion, the organisms disappear from the uterus
and uterine discharges and get localized into lymph nodes.

Main symptoms of the disease include abortion during third trimester of gestation and retention of
placenta in all the cases. The placenta appears dry, thickened, cracked, and covered by yellowish
exudates in the intercotyledonary areas. Cotyledons appear necrotic and may be covered with
exudates. In most of the cases the fetus is dead or dies soon after abortion. In male animals it
causes orchitis, epididymitis and degeneration of testes.

Diagnosis:

 History and symptoms

 Isolation of organism from uterus or uterine contents
 Milk Ring Test: used for diagnosis of brucellosis in the herd. In this test, antibodies
against Brucella are detected. One ml of milk is mixed with 1 drop (30 µl) of antigen and
incubated at 370C for one hour. In positive cases a blue ring is formed above white milk
column. In negative cases no ring is formed.
 Serum agglutination test.

Prevention and control:

 It can be prevented by maintaining proper hygienic conditions.

 Test and removal, segregation or slaughter.
 By vaccination with Brucella S-19 live vaccine. This is a calf hood vaccine. 2 ml of this
vaccine(Bruvax, Indian Immunological) is injected s/c at the age of 4-8 months in female
calves. Male and pregnant animals are not vaccinated. It may cause abortion in pregnant
animals and epididymitis in males.
 By vaccination with RB 51 vaccine. RB 51 strain of B. abortusis a genetically stable
mutant that lacks the polysaccharide O-side chain. As a result the vaccine is less virulent
and abortigenic than the strain S-19, but equally capable of producing an effective
immune reaction. Unlike strain S-19, RB 51 vaccine does not induce production of
antibodies that interfere with diagnostic testing. The calves are vaccinated between 4-12
months of age.

b). Mycoplasmosis: it is caused by Mycoplasma bovigenitalium and is transmitted by coitus or AI

with infected semen. The disease is also known as Granular or Nodular Vulvo-vaginitis. After
infection nodules of 1-3 mm diameter are formed in the vagina particularly around the clitoris.
 P a g e | 94

Animal feels pain during vaginal examination as well as after mating. It causes abortion during 5-
7th month of gestation. In male animal also the nodules are formed in prepuce around the glans
penis. These nodules are aggregate of lymphocytes. Disease can be prevented by avoiding natural
mating andadopting AI with disease free and antibiotic treated semen.

c). T.B.: It is caused by Mycobacterium bovis. It is mainly transmitted by ingestion of infected

feed or water. It may also be transmitted by natural mating. After infection, tuberculous lesions are
formed in uterus. From uterus yellowish discharge is noticed. After few days lesions are also
formed on outer side of uterine wall leading to severe adhesions of uterus. There is no treatment
and it can only be prevented by adopting the proper hygienic measures.

d) Mycotic abortions: It is caused by fungus like Mucor, Absidia, Rhizopusand Aspergillus. It

occurs mostly during winter season by consuming hay or silage because on silage and hay fungus
grows very well and the spores are either ingested or inhaled. At infection, it causes placentitis and
abortion during 4-9 months of gestation. On the skin of the aborted fetus „ring worm‟ lesions are
noticed. It can be diagnosed by demonstration of fungal hyphae from the fetus or placenta. It can
be treated by giving Mycostatin or Nystatin systemically.

e)Campylobacteriosis (Vibriosis): It is a venereal disease of cattle caused by the bacteria

Campylobacter foetis subspecies veneralis. Bulls serve as vector and permanent carrier for this
disease. They transmit the organism from female to female during coitus. Organism is found in
female genitalia, placenta and semen of the bull. The organism may also be transmitted by
artificial insemination of semen collected from the infected bull. Infection doesnot interfere with
fertilization but the endometritis it causes results in a uterine environment that is incompatible
with embryonic survival. Thus, when infection is introduced in susceptible herd, a dramatic
decrease in conception rate occurs. Early embryonic death occurring after maternal recognition of
pregnancy result in later return to estrus which occurs irregularly at more than 25 days after estrus.
A small number of animals abort typically at 2-4 months of gestation, although neither large
number of abortions nor abortion storms are characteristics of venereal Campylobacteriosis.
Placentitis is usually mild, the cotyledons may be haemorrhagic and the intercotyledonary area
becomes edematous.After experiencing serious infertility for about 6 months, a herd gradually
becomes immune and free from the infection. IgG based immunity develops slowly within the
uterus and infection is eliminated from the uterus, cow conceive and carry their fetus to term. Re-
infection of cows occurs if rebred with infected bulls result into less severe, infertility problem.
Maiden heifers bred to infected bulls will have low conception rates and irregular return to estrus
that are characteristic of Campylobacteriosis.

In young bulls(<3-4 years of age) in which crypts have not yet developed, infection
tends to be transient with transmission apparently relying on sexual contact with an infected cow
within a matter of minutes to days following the initial breeding of an infected cow.In cattle,
Campylobacter foetis subspecies veneralis and Campylobacter foetis subspecies fetus cause
Bovine Genital Campylobacteriosis and the disease is characterized by early embryonic death,
infertility and prolonged calving season. Infected cows may develop muco-purrulent discharge.
Bulls are asymptomatic. Diagnosis may require a direct culture of preputial smegma of bulls,
mucus secretions collected from infected females or the tissue collected from aborted fetus. AI
through semen of a disease free bull can control the disease. ELISA is the test of choice for the
diagnosis of Bovine Venereal Campylobacteriosis.
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f). Trichomoniasis: It is a classic venereal disease that is transmitted to cows from asymptomatic
carrier bulls during coitus. The causative organism is a flagellate protozoan, Tritrichomonas
(Formerly Trichomonas) fetus. T. fetus is an obligate venereal pathogen. Bulls become carrier by
serving an infected cow. The organism lives within the crypts and folds of penile integument and
preputial mucosa. The lack of these structures in younger bulls is probably the reason that the
organism is less able to establish itself in them. It causes early embryonic mortality after the
maternal recognition of pregnancy causing an irregularly extended return to the estrus, although
some exhibit normal or even short.However, infected cows will conceive to both infected and non-
infected services and eventually carry to term once immunity has developed. The disease is
characterized by early embryonic mortality, early abortion (2-4 months), pyometra and infertility.
RFM is very seldom observed.

Both Campylobacteriosis and Trichomoniasis are classical venereal diseases. Diagnosis of

Trichomoniasis is much easier than Campylobacteriosis. A positive diagnosis of Trichomoniasis
depends on a demonstration of live T. fetus organisms from the samples obtained from the female
cattle, preputial material of bulls or aborted fetal and placental tissues. The best source of material
is the fetal membranes or the organs of an aborted fetus especially the abomasums. Material
contaminated with faeces should be discarded because non-pathogenic trichomonad like
organisms may be present.

Treatment:

 Eliminating bulls from replacing natural service by AI.

 Trytaflavine (antiprotozoal), iodine based compounds, acriflavin and imidazole are used
for local application to penis and prepuce. Dimetanidazole can be given orally 50 mg/kg
for 5 days.

g). Neospora caninum: N. caninum is a recently recognized cyst forming, obligate, intra-cellular,
coccidian parasite closely related to Toxoplasma gondi (phylogenetically). It is known to cause
abortion or still birth in cattle, sheep, goat, horse and deer worldwide and neuromuscular paralysis
in neonatal calves. Thilsted and Dubey in 1989 for the first time reported Neospora associated
abortion in the cattle. In 1990s it became as a major cause of bovine abortions worldwide. Unlike
T. gondi it does not infect humans.

Life cycle: Dogs are definitive host where as domestic, wild ruminants and horses are
intermediate horses. Two types of transmission i.e . vertical transplacently from mother to fetus
and horizontally by ingestion of feed and water contaminated with Neospora oocysts. Ingestion of
colostrums contaminated with tachyzoites is also a mode of transmission. The disease is
characterized by fetal loss, retention of fetal membranes, fetal mummification etc. In majority
cases abortion occur in 5-6 month of gestation. Fetuses aborted due to N. caninum infection are
characteristically moderately to severely autolysed. At present no effective treatment for bovine
neosporosis. For prevention, dogs should not have access to calving areas, silage pits, feeding
areas and feed stores. Access to wild birds, rodents and other wild animals to feed should be
prevented to minimize the contamination of feed.
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h). Bovine viral diarrhoea (BVD): The BVD virus is a pestivirus which is related to Border
disease of sheep and classical swine fever. Mucosal disease is usually seen in younger animals (6
to 24 months). There are two genotypes of BVD virus: BVDV-1 which has worldwide distribution
and BVDV-2 which is largely restricted to USA. Disease associated with BVDV-2 is generally
much more severe than resulting from BVDV-1. Disease is characterized by pyrexia, water
diarrhoea, nasal discharge, buccal ulcerations and lameness. There are two main biotypes:
cytopathic and non-cytopathic strain. Infection with non-cytopathic strain in utero at 30-125 days
of gestation leads to birth of persistently infected calf. Persistently infected cows can transmit the
disease vertically through placental infection. Animal that are persistently affected or have acute
infection shed large amount of virus in occulo-nasal discharge, saliva, urine and faeces. Infection
in last trimester of pregnancy results in birth of a calf that is immune to disease. Infection in first
month of gestation results in early embryonic death and repeat breeding.There is low conception
rate with semen contaminated with BVD virus. Abortion can occur at any stage of gestation. Most
of the losses occur before the third trimester as the fetus increasingly able to mount an effective
immune response against the virus as gestation advances.From 5-6 month of gestation, there may
be abortion or birth of calves with congenital abnormalities of CNS and eye.

i). Infectious Bovine Rhinotracheitis(IBR): It is caused by Bovine herpes virus-1 (BHV-1). It

causes an acute respiratory disease of cattle. The disease of the genital system is known as
Infectious pustular vulvo-vaginitis (IPV), Vesicular venereal disease and coital vesicular
exanthema. BHV-1 causes both respiratory rather than genital form of the disease, although both
forms of the disease usually occur independently. BHV-1 also causes abortion, more commonly
after respiratory rather than genital form of the disease. . BHV-1 is also associated with infertility
in heifers and cows. Genital form is transmitted by coitus, contaminated bedding and mutual
licking. Abortion occurs from 4 month to term (most commonly between 4 to 8 months).
Spontaneous recovery of genital lesions will occur and therefore, no treatment is necessary.
Infected animals should be isolated. Heifers should be vaccinated with live attenuated vaccines at
six month of age followed by annual vaccination. Pregnant animals should be vaccinated with
killed vaccine.

j) Bovine Chlamydial Abortion/Foothill abortion/ Bovine epizootic abortion: Chlamydia

psittaci is a pathogen of both male and female genital tract. In bulls it causes orchitis, epididymitis
and believed to be involved in seminal vesiculitis syndrome. The organism is sometimes secreted
in semen of affected bulls. Characteristically, abortion occurs during the last trimester of gestation
without clinical signs. The abortions are seasonal, occurring 3-4 months or more after exposure to
ticks. Animals have to be less than 6 months pregnant at the time of exposure for abortion to
occur. The inter-cotyledonary areas of placenta are thickened, leathery and reddish white opaque
discoloration and edema is quite common. Once abortion has occurred animal is immune, so the
cattle that are at greatest risk are those calving for the first time and animals that have been moved
into tick infested region. Infection late in pregnancy can give rise to the birth of live and weak
calves. Lesions in aborted fetuses are characteristic and are used in its diagnosis. Abortuses are
typically not autolysed but have enlarged lymph nodes, spleen and liver and a reduction in the size
of the thymus. There may be petechial haemorrhages of the mouth, tongue, thymus and lymph
nodes. Histology is required to confirm the diagnosis: there is initial lymphoid hyperplasia which
may be followed by acute necrosis of lymphoid organs. Organisms can be cultured from aborted
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fetus and discharges. Giemsa stained smears or identification of elementary bodies or inclusions
are also useful. CFT and ELISA are also used to detect the infection.

k) Leptospirosis: Leptospirosis is an important zoonotic disease of cattle and other mammals and
is caused by pathogenic spirochaetes of the genus Leptospira. The pathogenic leptospires are
classified as Leptospira interrogans and non-pathogenic strains as L. biflexa. Infection can enter
through skin abrasions, mucus membranes of the eyes, mouth or nose. It can also be transmitted in
semen after natural service. After infection, there is latentperiod of 5-14 days followed by
bacteraemia for 4-5 days. After this animal shows immune response against Leptospirosis and the
organisms localize in tissues that are inaccessible to antibodies particularly renal tubules,
cotyledons and fetus. Organisms are secreted in urine and serve as source of infection. There may
be hepatitis, nephritis and hemolysis. The abortion is most commonly after 6 months of
pregnancy.

l) Listeriosis: Listeria monocytogenes is primarily a pathogen of the central nervous system in

sheep and cattle in which it causes encephalitis. Listeria monocytogenes is ubiquitous in the
environment, being present in the soil, sewage effluent, bedding and food stuffs; it persists as it is
resistant to the effects or drying, sunlight and extreme temperature. The source of infection in
cases of abortion is grass silage that is grossly contaminated with soil and has low dry matter
content or has undergone inadequate fermentation resulting in high-pH butyric silage. Organisms
gain entry through ingestion or mucus membranes of conjunctiva, or respiratory system. The
organism has predilection for placenta, causing placentitis, death of fetus and abortion. Abortions
are usually sporadic occurring towards end of pregnancy.

III. FUNCTIONAL INFERTILITY:

It affects individual animal and is not a problem in herd. It occurs mostly due to endocrinological
aberrations. It includes:

A. Cystic ovarian degeneration

B. Anestrous
C. Silent estrous
D. Delayed or anovulation
A. Cystic Ovarian Degeneration (COD): It is one of the major conditions causing infertility.
It is very common in dairy cattle than other species and is closely associated with milk
production in that it is seen more commonly in higher producing cattle. In cattle, the
incidence is 10-20%. It is high in lactating animals and more common during 2nd to 5th
lactation (4 to 6 years of age) and a higher incidence usually from 1 to 4 months after
calving with a peak between 15-45 days.Self-recovery occurs in 15-30% cases within 45
days of calving.The cyst can be of three type:
a) Follicular cyst/cystic degeneration of Graffian follicle
b) Luteal/Lutenized Cyst
c) Cystic corpus luteum
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a, b) Follicular and luteal cyst: Follicular cyst is characterized by presence of follicle or

follicle like structures which are usually more than 25 mm in diameter if single follicular
cyst or more than 17 mm in case of multiple follicular cysts and the thickness of follicular
wall is less than 3mm and the condition persist for more than 10 days. Luteal cyst is
characterized by diameter of corpus luteum more than 25 mm and persisting for more than
10 days with the thickness of luteal wall more than 3 mm. Follicular and luteal cyst are
anovulatory i.e. origin is from the follicle which fail to ovulate whereas cystic CL occur
after ovulation. Right ovary is more affected than left reflecting the relative activity of the
ovary.

Follicular cyst Luteal Cyst

1. Thin, soft and fluctuating 1. Always hard
2. May be multiple 2. Always single
3. Fluid in the follicular fluid is yellow 3. Fluid in the luteal cyst is brown in
colour
4. Animal is nymphomanic (Buller) 4. Animal is anestrous
5. Incidence is higher 5. Incidence is lower.
6. Thickness of follicular wall is less 6. Thickness of luteal wall is more than
than 3 mm 3 mm

Etiology:

i. It may be genetic/hereditary predisposition. It is more common in H.F. than other

dairy breeds.
ii. It occurs due to LH deficiency. Due to this, the dominant follicle fails to ovulate.
iii. Treatment of animal with excessive dose of estrogen hormone.
iv. Excessive feeding of plants having estrogenic activity e.g. beseeem. If a herd
shows more than 30% incidence of cystic ovaries and hereditary factor is not the
cause, estrogen in the feed should be suspected.
v. Hypothyroidism: If T4 level is less it causes alteration in the structure of granulosa
cells.

Symptoms:

Follicular cyst: Cows with follicular cysts are often nymphomaniacal (buller), i.e. displaying
excessive, prolonged signs of estrus and a shortened interval between successive heats. There is
edematous swelling of the vulva, frequent and copious discharge of clear mucus.The
nymphomanic cows are restless, nervous and bellow continuously. These cows are sexually very
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aggressive and behave like a bull. Animal affected with follicular cyst mount and stand to be
mounted. The animal may exhibit irregular or frequent estrus or sometimes animal may be
anestrous.There is decrease in appetite and milk yield. Animals‟ walking is increased. Due to
constant movement, mounting, and stirring up the other cows the buller animals lose weight.
After sometime, there is relaxation of sacrosciatic ligaments and in some animal dislocation of hip
joints or pelvic fractures may occur at the time of mounting. Due to long standing cases of
nymphomania, sinking of sacrosciatic ligaments and upward displacement of coccyx take place.
This elevated tail is known as Sterility hump. Once the sterility hump is developed, it persists even
after the treatment of cystic ovary.

On rectal examination, uterus is edematous, enlarged and flaccid. On vaginal

examination, the external os is slightly open and enlarged. Histologically, marked hyperplasia of
mucosa and cystic dilatation of endometrial glands take place and endometrium appears like
„Swiss Cheese‟. In some animals mucometra and hydrometra also develops. In the follicle
degenerative changes also take place. First, granulosa cell layer is affected and subsequently
degenerated. After that, the theca cell layers are affected. Cells of theca interna cell layer become
edematous and the intercellular space increases. After, sometime, the theca externa is also affected
and both the theca interna and externa appear like a single cell layer. No CL is palpable in the
ovaries with follicular cyst. A follicular cyst larger than 4 inches may indicate the presence of
granulosa cell tumor. Hydrometra or mucometra may develop following long standing case of
follicular activity and animals are more likely to be repeat breeders.

Luteal cyst: The luteal or luteinised cyst usually results in a cessation of cyclical activity; the
structure functions as a persistent corpus luteum. In the luteal cyst animal is always anestrous due
to continuous production of progesterone. If cows with luteinisedcysts are left untreated then a
proportion of them will become virilised i.e. these individuals will develop a masculine
conformation and will attempt to mount other cows, but unlike the nymphomaniacal cow they will
not stand to be mounted by other cows. In luteal cyst, the animal becomes very heavy and coarse.
The neck of animal becomes thick and heavy and a steer like appearance.This condition is known
as adrenal virilism. There are also the increased levels of 17-ketosteroids in the urine from the
adrenal gland.

Diagnosis of Follicular and luteal cyst:

i. Per-rectal examination.
ii. Ultrasonography: Follicular cyst: single(>25mm diame1ter) or multiple (>17 mm
diameter) anechoic structures. Thickness of follicular wall is less t1han 3 mm. Condition
persists for more than 10 days.
Luteal cyst: Single hyperechoic; thickness of follicular wall is more than 3 mm. Condition
persists for more than 10 days.
iii. P4 estimation: Progesterone concentration >2ng/ml in milk and >1ng/ml in serum or
plasma is indicative of luteal cyst whereas P4concentration in serum/plasma< 1 ng/ml is
indicative of follicular cyst.

Treatment of Follicular Cyst:

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i. Manual rupture of cyst; not recommended now because it may lead to ovario-bursal
adhesions.
ii. Casida (1944) first time treated the follicular cyst using sheep pituitary extract which was
predominant with LH. He used 5 IU of extract i/v. This extract is also called as vetrophin.
iii. As the condition is due to LH deficiency, so use Inj. Chorulon (hCG)3000-5000 IU i/m or
i/v. This hormone is a glyprotein hormone and in some cases it may cause anaphylactic
shock.
iv. GnRH analogues: 100µg
v. Progesterone: 50-100 mg i/m for 12-14 daysor PRID/CIDR/TRIU-B/ Ear implants for 10-
12 days. Due to negative feedback, it will stoprelease of gonadotrophins (FSH and LH).
And, upon withdrawal of treatment subsequent release of FSH and LH causes ovulation.

Treatment of Luteal Cyst:

i. PGF2α: Natural prostaglandin e.g Lutalyse: 25mg (5ml) i.m. or synthetic prostaglandin e.g.
Pragma 500µg (2 ml) i.m.
ii. Dexamethasone: 20 mg. Dexamethsone causes synthesis and release of PGF2α from the
uterus. But side effect includes, decrease in milk yield.

c).Cystic corpus luteum: It develops after ovulation i.e it is an ovulatory cyst. In this due to
unknown reasons a cavity is formed in the CL. On rectal examination, single CL appears similar
to normal CL but the cystic CL is slightly soft. If the animal is not inseminated, the cystic CL
regresses spontaneously. If animal is inseminated, it may or may not conceive. If size of cavity is
more than 10 mm, then animal will not conceive because less amount of progesterone is produced.
If it is less than 10 mm, then animal will conceive and cystic CL usually maintains the pregnancy
to term.Cystic CL often has a slightly fluctuating, soft consistency. This is the most common of
the cystic conditions affecting the bovine ovary with an incidence 2.5 times greater than cystic
follicles/follicular cysts. Cystic CL is not pathological as ovulation was normal and they do not
influence cycle length.

Prevention of Cystic Condition:

i. Since, the condition is genetic, therefore breed the female with the bull whose daughters
have less incidence of cystic ovary.
ii. Give a dose of GnRH 20 µg i/m on day 15-20post calving.

B. ANESTRUS
Anestrus is absence of overt signs of estrus whereas Acyclicity is absence of ovarian cyclic
activity. Long anestrus periods due to cessation of cyclic activity is known as true anestrus. In
other words, anestrus due to smooth ovaries or without palpable follicle and CL is known as
true anestrus. False anestrus may be physiological (lactational, gestational and open days) or
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pathological (due to pyometra and chronic metritis). Anestrus is the most common single cause
of infertility in buffaloes.
Broadly, anestrus is the failure of animal to exhibit estrus. It may be prepubertal or
postpartum. The animal fails to exhibit the signs of heat or estrus. It may be due to:
i. Congenital abnormalities like ovarian agenesis or hypoplasia, free martinism and
intersexuality.
ii. Retention of CL due to uterine pathological conditions of uterus like Pyometra, Chronic
Metritis and fetal mummification.
iii. Anestrus may be normal physiological after 45 days post parturition because this period
is required for normal involution of genitalia. Involution is the process by which uterus
attempts to return to its pre-pregnancy size and condition.The high levels of placental
and ovarian steroids i.e. estrogens and progesterone prevailingduring late gestation
exert a negative feedback effect on the hypothalamic–pituitary-gonadalaxis. After
parturition resumption of normal cyclic activity is based on re-establishment ofan
extremely coordinated hypothalamic–pituitary–ovarian–uterine axis.
iv. If the animal is pregnant, then animal will not show estrus. But sometimes in 5-10% of
cases the pregnant animal also exhibits estrus which is termed as gestational heat. It is
normally observed in first trimester of gestation.
v. Malnutrition: Due to poor nutrition in heifers there may be delayed puberty or ovarian
atrophy. Genitalia is poorly developed or infantile. In lactating animals it leads to small
and smooth ovaries.
vi. Hormonal Imbalance: If from the hypothalamus, pituitary and ovaries proper amount of
hormones are not produced may lead to anestrus. On rectal examination the genitalia is
properly developed but ovaries of such animals are smooth. No CL is present.
vii. Luteal cyst.
viii. Silent heat: In some cases, the signs are exhibited but not observed or poor expression
of heat symptoms may be there. The condition is termed as Silent heat. It is very
common in buffaloes particularly during summer. The animal is normal cyclic but
external signs (bellowing, frequent micturition) are not prominent because low level of
estrogen hormone which is responsible for external signs. There will be history of slight
vaginal discharge. On rectal examination either follicle or CL is observed on the ovary.
In unobserved heat, animal is normal cyclic and heat is observed at multiple of
normal estrous cycle days. e.g. 34, 36, 38, 40, 42, 46 etc. Animal may also be
unobserved in heat due to short heat duration i.e for less than 6 hours.
ix. Heat Stress: Due to heat stress the external signs of heat are poorly expressed and it is
common in buffaloes during summer. During summer there is more secretion of ACTH
and less secretion of LH.
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Treatment of anestrus:
i. Properly examine the animal and if animal is suffering from congenital abnormalities,
then cull the animal.
ii. If there is uterine pathology, treat it accordingly.
iii. Lugol‟s iodine paint:It is presumed that painting of Lugol‟s iodine on posterior part of
the cervix causes local irritation and brings about reflux stimulation at anterior pituitary
for secretion of gonadotrophins and consequently cyclicity. Lugol‟s iodine is an
irritating solution and Lugol‟s solution (0.5 to 1.0%) causes hyperemia (enhanced
circulation) of uterine mucosa resulting into degree of iodine absorption from uterus.
The absorbed iodine probably increases the metabolic rate of body through stimulating
the thyroid hormone secretion. Increased metabolic rate trigger the ovarian functions
by enhancing the energy utilization.
iv. In rest of the animals improve the nutritional status. For this supplement the ration with
50-60g of mineral mixture daily for 2-3 months. After this induce the estrus by the
hormonal treatment. For this we can give:
a). Estrogen: Give estrogen Progynon depot 10 mg i/m. Animal will come into heat within 2-3
days after injection. But in lactating animals it will cause drastic drop in milk yield. It should
never be given in lactating animals but can be used in heifers. In majority of cases the heat is
anovulatory, so no use of giving estrogen as animal will not conceive.
b). FSH: Follogon 1000 I.U.i/m. Animal will come into heat within 3 days. Disadvantages of
FSH/PMSG/eCG include:
 There will be twinning/triplets.
 In some animals it may lead to drop in milk yield.

c). Progestagens/Progesterone: Now a days progesterone therapy is widely used for estrus
induction. The progesterone may be natural or synthetic and used in various
protocols.

 Injection of progesterone: In this 50-100 mg of progesterone (Proluton depot/

Duraprogen) is administered for 12-14 days. After the last injection animal will
exhibit estrus within 3-5 days.
 MGA: It is synthetic progesterone known as Melengesterol acetate. Dose is 0.5-
1 mg in feed daily for 12-14 days. The estrus is exhibited within 5 days after last
feeding.
 Progesterone releasing intra-vaginal device (PRID): Device consist 1.55gm
progesterone, insert into vagina for 7-12 days and inject PGF2α one day before
or at the time of removal of device, animal will come into heat within 24-72
hours.
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 Controlled internal drug release device (CIDR): This is an intravaginal „T‟

shaped device/insert is impregnated with 1.38 grams of progesterone in elastic
silicone molded over a nylon spine. It is left into the vagina for 7-12 days and
PGF2αis injected at the time of removal of device or one day before the removal
of implant, animal will come into heat within 24-72 hours.
 TRIU-B (Virbac India): Each device comprises of 3 medicated rings (green
colour) containing Progesterone IP 186 mg each and one additional ring (pink
colour) with Progesterone IP 400 mg.
 Synchromate-B and Crestar ear implants: Synchromate-B contains 6mg
norgestomet in combination with an injection of 5mg estradiol valerate and 3mg
of norgestomet. Crestar ear implant contains 3mg norgestomet in combination
with an Injection of 5mg estradiol valerate and 3mg of norgestomet. The implant
is removed after 9-10 days and give better estrus synchronization within 24-72
hours after removal.

d). Ovsynch protocol/G-P-G regime: On day 0 inject GnRH analogue (Buserelin

acetate; Receptal) 20µg i/m. Inject PGF2α (Dianoprost tromethamine; Lutalyse)
25 mg i/m and again on day 9 give GnRH analogue (Buserelin acetate; Receptal)
20µg i/m. The heat is exhibited within 24-48 hours after the last GnRH injection.

 Acyclic animals respond much better to Ovsynch protocol than anestrus animals.

TREATMENT OF SILENT ESTRUS/UNOBSERVED ESTRUS:

Ask the owner about day of vaginal discharge and give PGF2α after 10-12 days
of vaginal discharge or palpate the CL and give PGF2α. Animal comes into heat
with 72 hours after PG. Treatment may also include double shot of PGF2α
injections at 10 days apart in cases of improper history of heat in cyclic
animals.

IV. MANAGEMENTAL : INFERTILITY DUE TO NUTRITIONAL DEFICIENCIES

 High yielding dairy cows require around 18-19% crude protein to sustain lactation.
 Cobalt: Deficiency occurs when diet contains less than 0.07mg/kg dry matter cobalt and is
due to failure of Vitamin B12 synthesis which is important factor for carbohydrate
metabolism. Deficiency causes anemia, inappetence, poor body condition, silent estrus,
irregular inter-estrus interval and poor pregnancy rates. Diagnosis can be confirmed by
measuring liver cobalt or vitamin B12 concentrations.
 Copper and molybdenum: Hypocuprosis can be either direct or indirect. Direct copper
deficiency due to an absolute deficiency of copper in diet is exceedingly uncommon. Most
cases of copper deficiency are indirect as a result of excessive levels of molybdenum, iron
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or sulphur in the diet which result in conversion of copper into insoluble, indigestible form
within the rumen. Copper deficiency is manifested as a reduction in first service
conception rates, impaired embryonic survival rates, poor estrus expression and reduction
in overall pregnancy rates. Impaired LH secretion has been implicated as the main means
by which these effects may be mediated.
 Iodine and goitrogens: The most common abnormalities associated with iodine deficiency
are embryonic death, abortion and still born or weak goitrous calves; loss of libido and
poor estrus behaviour. Disturbance of thyroid function can also be due to goitrogenic
substance present in lentils, soya bean, linseed and certain strains of white clover. The
dietary requirement for lactating cow is 0.05 ppm.
 Manganese: Manganese is a cofactor in a number of enzymes that are responsible for
gluconeogenesis and has a significant role as antioxidant. It is also involved in cholesterol
synthesis and hence steroidogenesis. Deficiency leads to delayed puberty, poor follicular
development, silent estrus, delayed ovulation and lowered conception rates.
 Calcium and Phosphorus: Deficiency of calcium and phosphorus leads to delayed
puberty, sub-estrus, irregular cycles, low conception rates, pre and post partum prolapse.
Deficiency of P also leads to post-partum haemoglobinurea. Blood P of less than 4 mg/dl
in animals at peak production are indicative of deficiency of P. The Ca-P ratio should be
between 1.5: 1 and 2: 1. Current NRC recommendations suggest that the Ca and P as 0.6%
and 0.4% of the ration on dry matter basis respectively.
 Selenium and Vitamin E: The primary role for which selenium is recognized clinically is
as a component of the cytosolic enzyme glutathione peroxidase (GPX). Cytosolic GPX
acts along with other antioxidants to prevent damage to cellular membranes induced by
free radicals arising from cellular metabolism of oxygen. The roles of selenium are
therefore wide spread throughout the body:
 Lipid peroxidation: Se along with active derivative of vitamin E, alpha-
tocopherol prevents lipid peroxidation of membranes, particularly of
skeletal and cardiac muscles.
 Arachidonic acid metabolism: GPX is involved in metabolism of
arachidonic acid into intermediates of prostaglandins.
 Phagocytic activity: Se deficiency mediated through GPX is associated with
reduced bacteriocidal activity of neutrophils, reduced proliferation of
lymphocytes and increased release of peroxides and macrophages.
 Iodothyronine 5-deiodinase: This is the enzyme which converts thyroxine
into the active thyroid hormone, tri-iodothyronine which is selenium
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containing. Growth may be retarded due to thyroid dysfunction because of

selenium deficiency.
The effects of selenium deficiency include retained fetal membranes,
delayed uterine involution, Metritis, cystic ovaries and impaired
fertilization. Deficiency of vitamin E is directly associated with embryonic
loss and affect rate of uterine involution through its role in immune system.
 Vitamin A and carotene: Vitamin A deficiency is associated with delayed
puberty, low conception and pregnancy rates.
 Zinc: Zinc is actively involved in a range of enzyme functions. The significance
appears more in males than females. Deficiency in males cause impaired
fertility associated with an alteration in latter stages of spermatogenesis as zinc
is involved as activator of enzymes involved in testosterone synthesis.
Deficiency in females cause decrease in fertility and abnormal estrus behaviour.

Epivag: Epivag is a specific bovine venereal disease causing epididymitis and vaginitis in cattle.
It causes vaginitis leading to mucopurulent discharge. Most infected cows fail to conceive and 15-
25% of animals become sterile because of the presence of lesions of uterine tubes such as
adhesions, ovario-bursal adhesions and hydrosalpinx. As a result of epivag infection parametritis
may also develop and adhesions may also extend upto entire pelvis and abdomen. In bulls, it
causes mild balanoposthitis (inflammation of glans and prepuce) and the infection is less severe
than IPV. After that, most of the bulls develop an induration of epididymis particularly at cauda
(tail) region and orchitis. Testicular degeneration, atrophy and fibrosis are sequent of the infection.
The causative agent is not known but considered a virus similar to herpes virus.

Catarrhal bovine vaginitis: Catarrhal bovine vaginitis is believed to be caused by enteric

cytopathic bovine orphan (ECBO) group but still remains to be proven. Transmission of the
disease is by venereal route, feacal contamination and licking the perineum of infected animals.
Infected cows have a profuse, post coital, non-odorous, mucoid vulval discharge. Vagina and
cervix are inflammed but without pustules as occur in IPV infection. There is no pyrexia. The
typical yellow gelatinous exudate accumulates in the vagina frequently in quantity from few to
hundred mililitres. Pregnancy rates are reduced. There are prolonged and irregular estruses
because of late embryonic death. In some animals, it may also lead to mummification, abortion
and still birth. In bulls it may lead to seminal vesiculitis and infertility.

INFERTILITY IN EWES AND DOES

Enzootic abortion of ewes: Enzootic abortion of ewes (EAE) is also known as ovine enzootic
abortion or kebbing. Infection is caused by Chlamydophila abortus (previously classified as
Chlamydia psittaci immunotype 1) which has no predilection for the pregnant uterus. It may also
infect goats, cattle, deer and humans and is the commonest cause of ovine abortion in UK. It is the
commonest cause of infectious goat abortion in USA. Major source of infection responsible for
80% of new outbreaks in clean flocks is by purchase of infected ewes of any age. Sheep to sheep
is the commonest route and lambing time is the greatest time of risk because infected ewes shed
large number of organisms at this time. Spread may also be by wild life e.g. foxes, gulls and
crows. C. abortus is not transmitted in the milk of infected ewes. However, lambs may acquire
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infection from uterine discharge on the teats. Tonsil and lymphoid tissue of the pharynx are the
primary site of infection with subsequent blood–borne spread to major organs and lymph nodes.
Abortion takes place in last 2 weeks of pregnancy in ewes and usually in last 4 weeks in does. The
macroscopic signs of placentitis are similar to that following Brucella abortus infection. The
intercotyledonary allantochorion is edematous, thickened and leathery; there is degeneration and
necrosis of the fetal cotyledons and thick yellow deposit on the chorion. Most of the aborted lambs
are well developed, fresh and without the autolytic changes. The lambs born alive are weak and
die within few days.

Toxoplasmosis in Ewes: Toxoplasma gondii causes early embryonic mortality, abortion, still
birth and weak offspring in many livestock species including sheep and has worldwide
distribution in animals and humans. It is the second most common cause of abortion in UK and
also in New Zealand. It is the commonest cause of infectious goat abortion in the UK. The
principal vector in spread of toxoplasmosis in the cat and its related wild species. Infective
Toxoplasma oocysts may survive upto 2 years on pasture, feed or bedding. Young cats which
become infected when they first begin to hunt. Toxoplasmas can also be passed in milk during
acute infections. Lateral spread within a flock from aborting ewes is likely to be relatively
unimportant. However, lambs born alive that survive from infected ewes can be congenitally
infected. Toxoplasma can be detected in the uterine caruncular septa 10 days after oocyst ingestion
and placental trophoblast cells after 10-15 days.
Following infection sheep are considered to remain persistently infected for life, to be immune to
the parasite and therefore unlikely to abort again with Toxoplasmosis. If ewes are in early
gestation i.e. 60-70 days, fetal resorption occurs commonly and ewes return to estrus or remain
barren. Infection in mid gestation results in abortion or mummification. In mummification only
one member of a set of twins or triplets may be involved. The gross appearance of placenta
especially cotyledons is fairly typical of the disease. Cotyledons are bright to dark red in colour
with multiple small white necrotic foci 1-3 mm in diameter (so called frosted strawberries). The
intercotyledonary areas of the allantochorion appear normal (unlike infection with Chlamydophila
abortus). T. gondii is of zoonotic importance, thus care should be taken in handling possibly
infected material.

Campylobacteriosis: Campylobacter is the third most common cause of abortion in ewes in UK

and the commonest cause in New Zealand. Both Campylobacter fetus fetus and Campylobacter
jejuni cause abortion in the ewes. Infected and symptomless animal excrete organism in faeces.
Campylobacter jejuni is mainly from a wild source and Campylobacter fetus fetus from carrier
sheep. Unlike cattle where the route of infection is mainly venereal, in sheep it is by ingestion and
mainly intestinal. Once abortion occurs, there is lateral spread to other susceptible pregnant ewes.
The only clinical sign of the disease is abortion usually in the last 6 weeks of the gestation. Lambs
born at full term may be born dead or in a weak condition.

Listeriosis: Two strains of Listeria produce disease in sheep are Listeria monocytogenes and
Listeria ivanovii. The disease may be presented in one or more of the following forms:
encephalitis; abortion; diarrohoea and septicaemia; keratoconjuctivitis, iritis and mastitis;
septicaemia and death in young lambs. Neurological signs characteristic of listerial encephalitis
are circling with evidence of unilateral facial paralysis, head tilting and turning. Abortions in
sheep are produced by both the Listeria monocytogenes and Listeria ivanovii and they can occur at
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any stage but most frequently in late pregnancy. The aborted fetus may be autolysed. Weakly
lambs are often born and grey/white miliary foci of necrosis may be seen in the fetal liver called as
„Saw-dust liver‟. The placental villi are necrotic and the chorion is covered with a brownish red
exudate and there is heavy brownish red exudate.
Encephalitis due to L. monocytogenes infection is quite common in goats and result in late
gestation abortion or still birth and rest of the clinical findings, pathogenesis is similar to ewes.

Border disease: The disease is due to infection with a pesti virus similar to bovine viral diarrhoea
in cattle. The new born lambs show tremors and a coarse fleece known as „Hairy Shakers‟ and are
generally weak with high mortality rate. The clinical signs are typical so called „Hairy Shakers‟ or
„Fuzzy lambs‟. Abortion can occur at any stage, however most common around 90 days with the
voiding of a brown, mummified or swollen anasarcous fetus.

Brucellosis: Sheep can be infected with both Brucella ovis and Brucella meletensis. B. meletensis
is primarily a disease of sheep and goats but can affect other species including man in which
disease is known as Malta fever. Transmission is by ingestion of contaminated feed and water. It
causes abortions, still births or weak lambs in late gestation. The placental lesions are similar to
Brucella infection in cattle. B. ovis mainly affect ram leading to epididymitis and subsequent
infertility or sterility.
B. meletensis is of zoonotic importance and causes abortion in late pregnancy, still births
or weakly kids in does. After first exposure the abortion may be in the form of storm. Few
abortions occur in other years and some does become sterile because of uterine lesions. Kids may
be infected by contaminated milk.

INFERTILITY IN MARE

Contagious Equine Metritis (CEM) in mare is caused by Taylorella equigenitalis which

commonly result in embryonic losses after initial infection. Carrier mares harbor the organisms in
vestibular area particularly the distoral fossa and sinuses.

Equine herpes virus (EHV) is the most common single cause of equine abortion. Abortion is
primarily caused by EHV-1 and occasionally EHV-4. EHV-1 is also capable of causing
respiratory disease (mostly in foals and yearlings), paralysis, neonatal foal disease and
uveitis/hypopyon. EHV-4 normally causes respiratory disease but occasionally cause abortion in
single mare. Transmission is by respiratory route. The majority (90%) of abortions occur within
60 days post infection, however the range from infection to abortion is 14-120 days. Abortion may
occur from 5th month to term but most commonly occur from 8-9 months to term. Typically, there
is sudden abortion without milk or udder development. Diagnosis of disease is by histopathology
of fetus. There are intra nuclear inclusion bodies in liver and lungs.

Equine viral arteritis: Equine viral arteritis (EVA) is a contagious disease of horses first
identified in 1953 and EVA outbreak was identified first time in UK in 1993.Outbreaks have been
attributed to the use of fresh cooled semen from a carrier stallion. Two main routes of EVA
transmission are venereal from semen of an infected stallion, and aerosol via respiratory secretions
of an acutely infected horse. Close or direct contact is required for aerosol transmission to occur.
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After an incubation of 7 days the EVA virus is secreted in all body secretions. After infection,
mares clear the infection and develop immunity. In stallion the virus persist indefinitely in
accessory glands and shed the virus in semen. Venereal route is the major cause of dissemination
of the virus. The virus is also transmitted through the use of fresh, chilled or frozen semen. The
classical clinical signs are an influenza like illness with pyrexia for 1-5 days, depression, nasal
discharge, anorexia, focal dermatitis; edema of limbs, ventral abdomen, scrotum, prepuce and
periorbital regions. The majority of abortions occur 23-57 days following exposure or 6-29 days
following the onset of fever in mare. Acute EVA can be confirmed by virus isolation from
nasopharyngeal swabs, heparinized blood samples, urine and semen samples.

Venereal endometritis in mare: In addition to opportunist pathogens, some bacteria e.g.

Taylorella equigenitalis (CEM organism), Klebseilla pneumoniae, and Pseudomonas aeruginosa
may be venereally transmitted by asymptomatic carriers of either sex. Prevotella fragilis,
anaerobic bacteria is isolated most frequently.
 Streptococcus zooepidemicus is the most commonly isolated bacterial species from acute
endometritis in mares particularly in initial stages. E. coli is the next most common isolate.

Equine coital Exanthema (ECE): ECE is a relatively benign venereal disease in both sexes
caused by equine herpes virus type-3. Transmission of organisms is by contact and during
gynaecological examinations. Once infected, horse remains carrier for life. Virus can remain
dormant until favorable conditions. Normally, following coitus signs develop after incubation of
4-7 days. Multiple vesicles develop on the vulvar mucosa and perineum leading to local irritation.
This causes rupture of vesicles resulting into small ulcers of 3-10 mm diameter which are painful
to touch. In the absence of secondary bacterial infection healing occurs in 10-14 days. There is
permanent loss of pigmentation at the site of the healed lesions. Pregnancy rates are not reduced
but mare is reluctant to allow mating because of pain associated with the ulcers. In stallion,
vesicles develop on the shaft of the penis and the prepuce, if they are severe, he may reluctant to
breed.

Trypanosoma equiperdum causes a venereal disease called dourine. It affects horse, mules, and
donkeys of either sex. The initial sign is a non-painful swelling of the external genitalia of both
stallion and mares. Mares show a vaginal discharge and stallions have paraphimosis (inability to
withdraw penis back into the preputial cavity). After some weeks, depigmented areas and urticaria
like raised plaques 2-10 cm in diameter appear over the body surface. The disease is characterized
by a low morbidity but a high mortality of 50-75%.

Endometrial cysts are of lymphatic origin and their occurrence may be associated with a
disruption of lymphatic function. The proportion of mare with endometrial cysts increases with
age. Mares over 11 years of age are more than 4 times as likely to have endometrial cysts as
younger mares and majority of mares over 17 years of age will have endometrial cysts. If intra-
uterine migration of embryo is inhibited by the cyst, the maternal recognition of pregnancy may
not occur. Diagnosis of endometrial cysts is best done by trans-rectal ultrasonography. In most of
the cases of endometrial cysts, no treatment is necessary if size is small. If size is large and
interferes embryonic mobility, then cyst need to be removed surgically. Laser surgery is an ideal
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method if equipment is available. Ligation and transaction of the stalk of pedunculated cyst is an
alternative. Thermocautery in conjunction with endoscopy is also possible.

INFERTILITY IN SOW

Cystic Ovarian Disease: Ovarian cysts are defined as pathological structures more than 10 mm in
diameter that are present in ovaries and are associated with the absence of normal cyclical ovarian
activity. Typically, ovarian cysts are multiple each being 20-30 mm in diameter, so that overall
diameter of the ovary can be upto 100 mm. The condition can be diagnosed ultrasonographically.
LH surge that triggers ovulation may not occur and the follicles continue to grow beyond the
normal size for ovulation of 7-9 mm in diameter. The impaired LH surge may be due to stress,
zearalenone toxicity, gonadotrophin administration in early diestrous or early induction of
ovulation after parturition. The incidence of ovarian cysts varies between 5-10%. Multiple ovarian
cysts may contain some luteal tissue, which if persists will inhibit subsequent LH secretion and
thus cyclical ovarian activity. Large multiple ovarian cysts generally severely compromise
fertility; single and smaller ovarian cysts may increase the rate of repeat breeding and result in
reduced litter size. The prognosis and response to any treatment are generally poor and culling of
the sow is advised.

Gilt disease: The disease is caused by Porcine parvo virus (PPV) and is a classical example of
viral pig disease mainly affecting the reproductive system. During embryonic period, the infection
causes embryonic death and resorption. In the fetal period if infection occurs before day 70 of
pregnancy, the fetus become mummified. As disease is called as Gilt disease, it is essential that
gilts are vaccinated twice before they are inseminated for the first time. PPV disease is wide
spread with herd prevalence in most countries exceeding 60%.

Porcine circo virus (PCV-2): Porcine circo virus infections are known to cause reproductive
disease in sow herds in a many countries. The virus infects the conceptuses following
transplacentation transmission. Embryonic/fetal death and loss of the whole litter may occur at any
stage of gestation. In addition to abortion, there is increase in number of still births, weak born
piglets or mummified fetuses. The oronasal route is the most common route of infection and the
horizontal transmission occur between pen mates and from one pen to other.

Porcine reproductive respiratory syndrome (PRRS): Porcine reproductive respiratory

syndrome is a viral disease that occurs in sows and gilts. The disease is characterized by severe
illness with pyrexia, inappetence, bronchopneumonia, and pleurisy. It affects reproductive system
and causes late term interruption of pregnancy, mummified fetuses, still births and weak born
piglets. Transplacental infection may occasionally also occur earlier during pregnancy. The
organisms are shed in saliva, nasal discharge, urine and faeces. In semen the virus may be shed
upto 43 days after exposure.

Aujeszky’s disease (pseudorabies): The disease is caused by herpes virus. The route of
transmission is inhalation or ingestion. It may also be transmitted by coitus. The disease is
characterized by nervous and respiratory signs associated a rise in temperature and often death in
young piglets. Infection in adults may result in still birth or abortion.

Leptospirosis: Leptospira pomona is the most widely recognized pathogen but serovars tarassovi,
bratislava and icterohaemorrhagiae may also be pathogenic in pigs. Site of predilection of the
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organism is kidney where they persist and reproduce and are secreted in urine upto 2 years.
Rodents are the reservoirs. In acute phase there is pyrexia and mild general symptoms while
chronic phase of disease is characterized by abortion during second half of the pregnancy.

Brucellosis: Brucellosis in pigs may not be highly pathogenic but its zoonotic nature makes it an
important infectious disease. The Brucella species in pig is B. suis, however B. neotome, B. ovis,
B. canis, B. abortus and B. meletensis may also occur. Infection is usually acquired by oronasal or
genital route. Abortion may occur at any stage of pregnancy. As sows usually eliminate the
bacteria within 30 days after infection, a period of reproductive rest of 2 estrous cycle (42 days)
may be enough to eliminate the infection. Other signs of the disease include sub-fertility,
orchitis, posterior paralysis and lameness. Boars are permanent carrier.

CYSTIC ENDOMETRIAL HYPERPLASIA-PYOMETRA COMPLEX

Pyometra is the accumulation of purulent material within the uterine lumen of intact
bitches, typically occurring during or immediately following a period of progesterone
dominance. Sub-acute endometritis is followed by cystic endometrial hyperplasia (CEH)
which then predisposes bitches to develop pyometra. It is common to see pyometra defined
as the endometritis/cystic endometrial hyperplasia/pyometra complex (CEH/P). Pyometra is
classically a disease of the diestrual bitch and can be classified as open-cervix or closed-
cervix, with the latter being a medical emergency requiring rapid intervention to prevent
subsequent sepsis and potential patient death. Mucometra, hematometra, or hydrometra are
thought to occur with CEH, and the character of the intrauterine fluid differs from pyometra
in that they are classically all sterile and are the fluid is sero-mucous, bloody, or serous,
respectively.
ETIOLOGY AND PATHOGENESIS:
Hormonal therapies that include either progestins for estrus suppression or estrogens
for estrus induction or pregnancy termination may cause the development of pyometra in
young bitches. Young bitches with anatomic abnormalities of the vagina and vestibule, such
as strictures and septums, may be predisposed to develop these conditions. Cystic endometrial
hyperplasia develops after repeated progestational stimulation during the luteal phase of the
estrous cycle. Although CEH is generally thought to predispose the bitch to develop pyometra,
the two conditions can develop independently of one another. Furthermore, CEH is also
associated with mucometra, as CEH can result in endometrial thickening with the
accumulation of viscid fluid in the uterus. The main difference between mucometra and
pyometra is that with mucometra the fluid accumulating in the uterus is sterile and with
pyometra, it is contaminated with bacteria. During diestrus, a time of progesterone dominance
in the bitch, progesterone increases endometrial gland secretory activity, increases
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endometrial proliferation, decreases myometrial contractility, and causes closure of the

cervix. These effects are cumulative after repeated estrous cycles, explaining the increased
incidence in middle-aged to older bitches. Estrogen stimulation prior to progesterone
dominance is also a component of the pathogenesis.
The combination of reduced local immunity in progesterone dominance in diestrus and
favorable uterine conditions for pathogens (increased glandular secretions and decreased
myometrial activity) make bacterial colonization more likely during this phase. The most
common bacteria isolated in cases of pyometra is Escherichia coli w h i c h is also usually
found in the feces of affected bitches,
although Staphylococcus,Streptococcus, Pseudomonas, Proteus spp, and other bacteria also
have been recovered. Bacterial contamination of the uterus likely occurs prior to diestrus
when the cervix is open, and in cases of CEH, the bacteria cannot be cleared prior to the
luteal phase, leaving opportunistic organisms in a prime environment for c o l o n i z a t i o n
and proliferation. E. coli is a particularly dangerous organism in cases of pyometra, due to
endotoxin release which may result in septic shock.

CLINICAL SIGNS

Clinical signs of pyometra in dogs depend primarily on whether the cervix is

patent enough to allow drainage of purulent fluid. The most common clinical finding in bitches
with open-cervix pyometra is a foul smelling, sanguinous to mucopurulent vaginal discharge.
Bitches with open-cervix pyometra are generally less systemically ill than bitches with closed-
cervix pyometra and early in the course of the disease affected bitches may show no clinical signs
other than vaginal discharge. Additional clinical findings may include lethargy, depression,
inappetance/anorexia, polyuria, polydipsia, vomiting, and diarrhea.
In contrast, bitches with closed-cervix pyometra are generally very ill at presentation, with
marked clinical signs of depression, lethargy, polyuria, polydipsia, vomiting, diarrhea, and
possibly abdominal distension. Affected bitches are often dehydrated and septicemic, toxemic,
and in shock. Fever may be present in bitches with pyometra, but those with toxemia may
actually be hypothermic. There is typically no evidence of vulvar discharge. In open cervix-most
often the consistency of vulval discharge is thin and light chocolate brown in colour and has a
characterstic odour.
Pyometra causes varying degrees of systemic illness, but mucometra typically does not
cause obvious clinical signs. Both entities can cause palpable uterine enlargement due to fluid
accumulation. In pyometra, the size of the uterus is inversely proportional to the degree of
cervical patency. Death may be due to toxemia aone or it may be associated with peritonitis
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due to rupture of uterus.

DIAGNOSTIC FINDINGS

Clinical pathology: Cytologic examination of vaginal discharge is an initial and very helpful tool
in diagnosing canine pyometra and in differentiating open-cervix pyometra from mucometra.
Neutrophils which are often degenerative and present in large numbers are frequently seen on
cytologic examination of vaginal discharge with pyometra. With mucometra, cytology may reveal
lesser numbers of neutrophils, with or without degenerative changes, red blood cells, endometrial
cells (usually with foamy cytoplasm), and varying amounts of amorphous debris. With hydrometra,
scant red blood cells and white blood cells, moderate numbers of endometrial cells, minimal mucus
and amorphous debris are evident. With hematometra, red blood cells are the predominant cell
type, with scant white blood cells and minimal to moderate mucus, scant endometrial cells and
minimal to moderate amorphous debris present.

A common pathologic clinical finding in bitches with pyometra is a peripheral

leukocytosis, which is more pronounced in closed-cervix pyometra. When a differential cell
count is performed, a left shift is also a common finding. A normocytic, normochromic anemia
may be seen in cases of pyometra, with packed cell volumes ranging from 21 to 48%.
Abnormalities in serum chemistry include azotemia, hypergammaglobulinemia, and
hypoalbuminemia. Metabolic acidosis is a common finding. Urinalysis findings are less
consistent, as dehydration affects urine specific gravity. Decreased urine specific gravity has
been reported in approximately 20% of cases of pyometra. Specific renal abnormalities have
been described in pyometra including the decreased ability of the renal tubules to concentrate
urine as a result of the effects of endotoxin, which accounts for both the decreased specific
gravity and the clinical signs of polyuria and polydipsia. Proteinuria may be observed. With
mucometra, the serum chemistry, leukogram and urinalysis are typically without abnormality.

Diagnostic imaging: The diagnosis of pyometra is best made with the aid of ultrasonography and
findings typically include an enlarged uterus with convoluted, tubular horns filled with anechoic to
hypoechoic fluid. The luminal contents are usually homogenous, but the contents may also be
echodense with slow, swirling patterns. A thickened endometrium with cystic structures is
diagnostic for CEH, with or without pyometra. Endometrial edema may be present. Mucometra or
hematometra are suspected if uterine luminal contents are echodence and hydrometra is suspected
it luminal contents are anechoic in combination with a lack of clinical signs consistent with
pyometra. Radiography may also be used as an aid in diagnosing pyometra in the bitch, but can be
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frequently inconclusive. The normal non-pregnant and early pregnant uterus is of soft- tissue or
fluid radiopacity.

Fig.: Cystic Endometrial Hyperplasia

Fig: Ultrasonograph of CEH in a bitch

TREATMENT
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Ovariohysterectomy is the treatment of choice for pyometra. Medical management could be

considered if preserving the reproductive potential of the bitch or queen is desired. Fluids (IV) and
broad-spectrum, bactericidal antibiotics should be administered. Fluid, electrolyte, and acid-base
imbalances should be corrected as quickly as possible, before ovariohysterectomy is performed.
The bacterial infection is responsible for the illness and will not resolve until the uterine exudate is
removed. Oral antibiotics (based on the results of the culture and sensitivity) should be continued
for 7–10 days after surgery.

A combination of prostaglandin and prolactin inhibitor, cabergoline can be found to be successful

for treating both open and closed cervix pyometra. In all the cases therapy should be combined
with appropriate broad spectrum antimicrobial agents and intra venous fluid therapy.

Other antiprogestins (eg, aglepristone) are available in some countries. Clinicians using
aglepristone report virtually no adverse effects as compared with prostaglandins. Aglepristone is
also used to treat bitches with closed cervix pyometra. A dosage of 10 mg/kg given on days 1, 2,
and 8 in 15 bitches with closed pyometra cases can open cervix in 1-2 days.

SPLIT HEATS

Split heats are observed in young pubertal bitches but can occur at any time in life.
In split heat, follicles develop, produce estrogen and the bitch exhibits all signs of
proestrus. However, ovulations do not occur. Four to 10 weeks later, the bitch once again
enters into estrus. The second half of split heat is always an ovulatory heat. Diagnosis is by
vaginal cytology and serum progesterone estimations.

The incidence of spit heat in bitches is more common at the onset of

puberty and with old age. Spit heat consists of two phases. In the first phase of a split heat
there is normal development of the ovarian follicles with secretion of adequate estrogen
that causes typical signs of proestrus such as vulvar swelling, vaginal bleeding, attraction
of males, some behavior changes and rarely, breeding. However, these follicles instead of
ovulation regress with all signs of proestrus/estrus dissipating. The second phase which
occurs two to 10 weeks later is characterised by the development of a new wave of
follicular growth and secretion of enough estrogen for signs of proestrus to reappear.
Vaginal bleeding is once again seen. However, the second phase of split heat is a “true”
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heat where the bitch is typically fertile with ovulations occurring normally. Breeding in the
true cycle usually results in production of a normal litter. The proestrual bleeding seen
during the second follicular wave may be interpreted by most owners as bleeding seen with
abortion or resorption of fetuses.

Diagnosis is by vaginal cytology because each follicular wave is associated with

estrogen secretion and typical estrogen induced proestrus changes. The diagnosis can be
further supported with serial serum progesterone assessments. Usually, progesterone
concentrations never exceed 2 ng/ml during the split heat, indicating that ovulations never
took place. The progesterone concentration may rise during estrus then the progesterone
concentration returns to basal levels. Such heats are not considered worrisome. No
association exists between split heats and later infertility, nor between split heats and
ovarian or uterine disorders. Recognition of split heats does require close supervision of
the bitch and good communication between owner and veterinarian. The possibility of split
heats is one reason for not treating any bitch for shortened interoestrous intervals until she
is at least 3 year of age.

SPECIFIC INFECTIONS OF BITCHES

Brucella canis

B. canis is a Gram-positive bacterium that can produce abortion and infertility. It is the
only bacterium known to be a specific cause of infertility in the bitch. B. canis can be
transmitted in several ways, including contact with aborted fetal or placental tissue, contact
with the vaginal discharge of infected bitches, venereal transmission and congenital
infection. The most common method of infection is venereal. Abortion occurs most
commonly between days 45 and 55 of pregnancy; however, there may be early fetal
resorption, or the birth of stillborn or more rarely weak pups.

Toxoplasma gondii

T. gondii infection causes abortion, premature birth, stillbirth and neonatal death. Surviving
infected pups may carry the infection. The public health consequences of Toxoplasma infection
should be considered whenever it is diagnosed.

Canine herpesvirus

Canine herpesvirus in adult dogs generally produces a few mild signs limited to the
respiratory or genital tract. However, the virus may cause genital lesions in the bitch that
may be associated with infertility, abortion and stillbirths. It appears that infection of the
pregnant bitch results in the production of placental lesion and the infection of the fetuses.
The infected placentae are macroscopically underdeveloped, and possess small grayish
white foci characterized by focal degeneration, necrosis and the presence of eosinophilic
intranuclear inclusion bodies. Pups may become infected at birth, during passage through
the vagina, and subsequently die with characteristic widespread histological necrotizing
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lesions. Pups that survive the illness may show persistent neurological disorders. In the
pups, the disease is rapidly fatal and treatment is often unrewarding; symptomatic therapy
is all that is available since specific antiviral agents are not efficacious. Fetal
mummification is characteristic of canine herpes virus (CHV) infection in bitches.

Canine adenovirus

It is well established that infection with canine adenovirus during pregnancy can
result in the birth of dead or weak pups that die within a few days of whelping. In most
cases, however, the virus is ingested and causes neonatal mortality. Carrier bitches may
therefore act as a source of infection for pups.

Canine distemper virus

Experimental exposure of pregnant bitches to canine distemper virus was found to

produce either clinical illness in the bitch with subsequent abortion, or subclinical infection
of the bitch and the birth of clinically affected pups. This provides evidence for
transplacental transmission, although the frequency of this under natural conditions is
unknown.

Canine parvovirus

Canine parvovirus has been implicated by some breeders as a cause of infertility in their
kennels. Canine parvovirus may cause an acute generalized infection in pups less than 2
weeks of age, which can occur as a consequence of uterine infection or as a result of
exposure to the virus soon after birth.

SPECIFIC INFECTIONS OF QUEEN

Feline Leukaemia Virus (FeLV)

Feline Leukaemia Virus (FeLV) has been implicated in a variety of clinical syndromes
including infertility, embryonic resorption and abortion. FeLV is believed to be the single
most common cause of infertility in the queen. Fetal resorption is seen frequently, although
abortion and the birth of permanently infected kittens also occur. The aetiology of the
reproductive disease is uncertain, and whilst it is known that the virus may cross the
placenta, one possibility is that secondary bacterial infections occur because of FeLV
induced immunosuppression. Diagnosis of FeLV may be achieved by virus isolation.

Feline Herpesvirus

Feline herpesvirus may result in abortion during the 5th or 6th week of gestation. Lesions
may be found within the uterus; however, placental lesions have only been demonstrated
following experimental infection. In the naturally occurring disease, abortions are thought
to be the result of a non-specific reaction to the infection Transmission of the virus occurs
via the respiratory tract, with up to 80% of cats remaining as chronic carriers. The
diagnosis of herpes virus infection is based upon the clinical signs and the isolation of
virus.

Feline Panleucopenia Virus

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Feline panleucopenia virus is transmitted by direct contact with salvia, faeces and
urine. Infection of pregnant queens may result in abortion, stillbirths, neonatal deaths and
fetal cerebellar hypoplasia. These effects are the result of transplacental infection leading
to fetal death and resorption in early pregnancy and cerebellar hypoplasia when infection
occurs from the middle third of pregnancy onwards. Diagnosis may be made on the basis
of the clinical signs, histopathological findings, virus isolation and paired serum samples
that demonstrate a rising antibody titre.

Feline Infectious Peritonitis Virus

Feline infectious peritonitis virus has been implicated as a cause of infertility, stillbirths,
endometritis, resorption and abortion, chronic upper respiratory tract disease and fading
kitten syndrome. Queens are not always ill and may suffer resorption of abortion which is
unnoticed. Abortion generally occurs during the last 2 weeks of pregnancy. Diagnosis is
made by serological and pathological investigation.

Toxoplasmosis

Toxoplasmosis has been incriminated as a rare cause of abortion and congenital

infection of cats. Serological screening is necessary to demonstrate the role of this
protozoan in cases of abortion.

Chlamydophila felis

There is evidence that the feline strain of Chlamydophila felis causes abortion in
the queen. The mode of transmission has not been elucidated, although the organism has
been isolated from the genital tract of infected cats and there is circumstantial evidence
associating infection with reproductive disease as well as abortion. Diagnosis is possible
by demonstrating high antibody titres. It is difficult to confirm whether the isolation of this
organism indicates its role in an abortion, since it may simply be an opportunistic
bacterium.

Note: Fetal mummification is very common in the pig and is a particular characteristic of
infection with the SMEDI virus. (S- Still birth, M- mummification, ED- Embryonic death,
I- Infertility). It is also seen in large litters as a consequences of uterine overcrowding and
placental insufficiency similar to bitches and queen.

EVALUATION OF DAIRY HERD FERTILITY

Regular, accurate evaluation of fertility status of the dairy herd is an essential part of
control programme. In an „all-year-round calving‟ herd it should be done at least twice a year. In
seasonally breeding calving herd it should be done at times appropriate to the desired calving
pattern. The minimum information required is identity of the cows, last calving date, first and
subsequent service or insemination dates, confirmation of pregnancy and dates when cows are
culled or left the herd.

The following measurements of fertility can be made:

1. NON RETURN RATE TO FIRST INSEMINATION: This is percentage of heifers/cows in

a particular over a specified period of time that has not been presented for a repeat insemination
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within a specified period of time. This period is usually 30-60 days or 49 days. This is used,
particularly in artificial insemination centres, to monitor the fertility of bulls and the
performance of inseminators.
2. CALVING INTERVAL OR CALVING INDEX (CI): The calving interval is the interval in
days for an individual cow between successive calvings; the calving index is the mean calving
interval of all the cows in a herd at a specific point in time, calculated retrospectively from their
most recent calving date. These two measurements have been used traditionally as a measure of
fertility, since they indicate how closely the individual cow or herd approximates to the
accepted optimum of 365 days.
The disadvantages of these measurements are that they are historical in that they are
calculated retrospectively; furthermore, the calving index can give an overoptimistic
assessment of fertility when many of the cows that fail to become pregnant are culled.

The calving interval (or index CI) is the sum of two components, (a) the interval from the
last calving date to the date of conception and (b) the length of gestation, thus:

CI = a + b

Therefore

CI = 85 days + 280 days = 365 days

3. CALVING TO CONCEPTION INTERVAL (CCI) is calculated by counting the number of

days from calving to the service which resulted in pregnancy (effective service); this is usually
the last recorded service date. The CCI is a useful measurement of fertility but requires a
positive diagnosis of pregnancy to be made. It is influenced by two factors; how soon after
calving the cows are served or inseminated and how readily they become pregnant when they
have been served.

The CCI can be expressed as:

Mean CCI = c +d

Where „c‟ is the mean calving to first service interval and „d‟ is the mean first service to
conception interval, therefore

Therefore, Mean CCI = 65 days + 20 days = 85 days.

4. OPEN DAYS/VOLUNTARY WAITING PERIOD/SERVICE PERIOD

This is defined as the interval in days from calving to the subsequent effective service date
of those cows that conceive, and from calving to culling or death for those cows that did not
conceive.

Numerically, it will always be greater than the mean CCI unless all cows that are served
conceive in which case it would be the same.
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Days open (%) = (Total service period in days in all lactations /Total herd life in days) x 100

5. CALVING TO FIRST SERVICE INTERVAL

In the case of a herd that calves all the year round a mean value of 65 days should result in a
mean CCI of 85 days. The factors that influence the calving to first service interval are:

i. Breeding policy of the farm: Although some cows return to estrus after calving as early as
2-3 weeks, but they should not be served before 45 days, and in the case of first calving,
high-yielding cows and those that have had dystocia and problems during the puerperium
slightly longer time should elapse and should not be served before 60 days. Thus, in a
seasonal calving herd, those that calve early in the season will have their first service
delayed and, for those that calve late, it may be necessary to advance the date of first
service thereby tightening the calving pattern.
ii. Delayed return of cyclical activity after calving, i.e. true anestrus
iii. Failure to detect estrus in those cows that have resumed normal cyclical activity.
iv. Factors (ii) and (iii) can be improved by ensuring that cows have returned to cyclical
activity postpartum. This can be done by regular and routine examination of those cows,
per rectum, that have failed to be seen in estrus by 42 days postpartum and by the use of
milk progesterone assays and ultrasonography.
v. Detection of estrus depends upon the herdsman knowing the true signs of estrus, having a
regular routine, recording the events and using estrus detection aids.

6. OVERALL PREGNANCY RATE

This (originally called the overall conception rate) is the number of services given to a
defined group of cows or heifers, over a specified period of time which result in a diagnosed
pregnancy not less than 42 days after service; the figure is expressed as a percentage of the total
number of all services and should include culled cows. The method of pregnancy diagnosis
should be specified. The first service pregnancy rate is usually calculated separately and
obviously refers to first services only. Thus, in a 12 month period, if 100 cows receive 180
services, of which 90 resulted in a confirmed pregnancy, the overall pregnancy rate would be
50 per cent. The pregnancy rate to first service and overall pregnancy rate are very useful
measures of fertility; the overall pregnancy rate is used to calculate the reproductive efficiency
of the herd. The rates for the first service are usually slightly higher than those for all services
because the latter group will include those cows that may be sterile and receive many services
before they are culled. In order to identify the influence of management changes, particularly
nutrition, it is worthwhile calculating these two parameters on a monthly basis provided that
there is a minimum of 10 services per month. The pregnancy rate is influenced by:

 The correct timing of artificial insemination, which will be dependent particularly on

the accuracy of estrus detection.
 Correct artificial insemination technique, handling and storage of semen.
 Good fertility of the bull if natural service is used, and the absence of venereal disease.
 Adequate nutritional status of cows and heifers at the time of service and afterwards.
 Complete uterine involution and absence of uterine infection.

7. OESTRUS DETECTION RATE

Improving the detection of estrus has a much greater influence upon reducing the calving
to conception interval than improving the pregnancy rates. This enables herdsmen to anticipate
the time of a subsequent estrus and thus improves the detection rate. It also enables the early
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detection of acyclic cows. It is possible to estimate the estrus detection rate but it is important
to stress that it is an estimate and not an accurate measurement. A number of different methods
are used and they all have some measure of inaccuracy. One method is to determine the number
of supposed missed estrous periods. This an interval of 36-48 days suggests that one estrus has
been missed and an interval of 54-72 days suggests that two have been missed, although this
latter range is fairly wide and can lead to errors. The percentage of estrus detection rate (ODR)
is calculated thus:

Oestrus detection rate (%) = (No. of interservice interval recorded / (No. of interservice
interval recorded + No. of missed estrous period) ) x 100

Or (21/mean interservice intervals) x100

Efficiency of oestrus detection (%) = (No. of estrus detected / Total No. of estruses) x 100

Accuracy of oestrus detection (%) = (No. of estrus detected / (No. of estrus detected + No. of
false estrus detection)) x 100

8. FIRST SERVICE SUBMISSION RATE

It is the measure how quickly the cows are served after the end of voluntary waiting
period/service period/open days and in seasonal breeding herd after the start of breeding period.
It is defined as the number of cows/heifers served within 21 or 24 days period expressed as a
percentage of those eligible to breed at the start of the 21- or 24 days period. It has been shown
that there is a good correlation between the physical state of the uterus, as determined by rectal
palpation and the amount of mucopurulent discharge and the regeneration of the endometrium.
Heifers, and cows yielding more than 40 litres per day, should not be served before 50 days
postpartum.

The submission rate is influenced by: (i) the time interval to the resumption of normal
cyclical activity after calving, and (ii) the detection of estrus in those cows that have resumed
normal cyclical activity, and their presentation for service or artificial insemination. A good
submission rate is 80 per cent. A relatively simple method of obtaining a fairly accurate
measurement is to list all cows that are ready for service (at or beyond the earliest service date
of 45 days since calving) at the start of each 21 or 24 day service period. At the end of this
period identify all those that have been served. The percentage submission rate is calculated
thus:

Submission rate (%) = (No. of cows or heifers served within a 21 days period / No. of cows or
heifers that are at or beyond the start of the 21 days period) x 100

Another method is to list all cows chronologically in order of the calving date. Add 21
days to the earliest date on or after which they are ready for service, i.e. 45 + 21 (24) = 66 (69)
days. Thus, every cow should be served before the target date of 66 or 69 days postpartum. The
submission rate is calculated as:

(No of cows served on or before the target date / No. of cows that should have been
served on or before the target date) X 100
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9. REPRODUCTIVE EFFICIENCY

Attempts have been made to calculate a single index that provides an overall measurement
of fertility and takes into account many different parameters. One such measurement is the
reproductive efficiency (RE) of the herd; it is calculated as:

RE = (Submission Rate X Overall Pregnancy Rate) / 100

 Thus if the submission rate is high, i.e. 80 % and the overall pregnancy rate is good, i.e. 55 %,
then the RE is 44.
 If S.R is 70% and O.P.R. is 50%, then RE is 35

10. CULLING RATE

One method of achieving a CI around 365 days is by culling those cows that are
slow to get in calf. This is rarely cost-effective because it will be necessary to replace the
culled cow with a heifer. The purchase price or the cost of rearing such a replacement is
much greater than the price obtained for the cull. Overall culling figures for infertility
should not exceed 5per cent; thus 95per cent of the cows that calve and are served should
become pregnant again.

11. FERTILITY FACTOR(FF):

FF is obtained by the following formula:

ODR x OPR/100
Where ODR is Oestrus detection rate and OPR is Overall pregnancy rate.
If ODR is 60% and the OPR is 50%, then FF is 50 x 60/100 = 30.

Repeat Breeding Syndrome

A normal cyclic female animal with apparently normal genitalia mated in three or more
consecutive estruses with fertile bull or inseminated artificially with fertile semen if fails to
conceive is called as Repeater/ Cyclic non-breeder and the condition is termed as Repeat
Breeding. As the condition is multifactorial in origin, therefore termed as Repeat Breeding
Syndrome.

A repeat breeder/Cyclic non-breeder cow is one -

• That has been bred 3 or more times but not conceived
• Has normal estrous cycle
• Is free from palpable abnormality
• Has no abnormal vaginal discharge
• Has calved atleast once and
• Is less than 10 years old

The etiological factors may be: Male, female and managemental factors

1) Male factors:
i. Infertile bull
ii. High sperm abnormalities
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iii. Low sperm motility

Check the semen quality and use only fertile quality semen.
2) Female factors:
i. Delayed ovulation: Diagnosis is difficult. Treat with GnRH analogue
(Receptal/Gynarich 2.5 ml i.m) or hCG (1500 IU i.m) at the time of insemination
and repeat insemination on the next day.
ii. Anovulation: Diagnosis depends on ovarian palpation or transrectal
ultrasonography 7-10 days after estrus to demonstrate failure of ovulation by
absence of a CL. The treatment of the condition can be done by hCG or GnRH
analogue.
iii. Fertilization failure: it may be due to defective ovum or aged ovum. In
immunological problems, sexual rest for 2-3 months and breeding is to be avoided.
Change of bull or heterologous semen doses for AI (from two different
breeds/bulls).
iv. Luteal insufficiency: 500 mg of progesterone depot on 5th , 10th and 20th day after
AI or 1000-1500 IU of hCG/ 50g GnRH (10 g of GnRH analogue) on 6th day of
cycle. Alternatively, give hCG or GnRH analogue 11-13 days after breeding. The
rational for this approach is to induce accessory CL or luteinized follicles or to
augment the progesterone secretion.
v. Endometritis: Diagnosis of endometritis can be done by white side test and can be
treated by intrauterine therapy. Cultural sensitivity of estrus discharge can be done
to find out the microorganism and the sensitive antibiotic.
vi. Hydrosalpinx: Bilateral hydrosalpinx makes the animal sterile. Unilateral
hydrosalpinx ipsilateral to ovary undergoing ovulation lead to repeat breeding.
vii. Ovario-bursal adhesions: Bilateral ovario-bursal adhesions make the animal sterile.
Unilateral ovario-bursal adhesions ipsilateral to ovary undergoing ovulation lead to
repeat breeding.
viii. Tubal blockage/Occluded oviducts: Tubal blockage can be diagnosed by PSP,
Indigocarmine and Starch test. Bilateral blockage makes the animal sterile and
unilateral blockage ipsilateral to ovary undergoing ovulation result in to repeat
breeding.
ix. Early/Late embryonic death: In case of early embryonic death before maternal
recognition of pregnancy or upto 10-12 days post mating/AI, the animal is normal
cyclic at regular interval. If embryonic death is after the aforesaid period i.e. late,
the cycle length is increased.
x. Anatomical defects: In nulliparous animals look for segmental aplasia. Cull the
animal with anatomical defects.
xi. Follicular cyst: In follicular cyst animal is nymphomanic and there is anovulation,
therefore animal is repeat breeding. Treat the follicular cyst with GnRH/hCG or
progesterone. The dose of GnRH is 100 g (20 g of GnRH analogue), Chorulon
(hCG) 3000 IU i/m or i/v. This hormone is a protein hormone and in some cases it
may cause anaphylactic shock. Alternatively, progesterone: 50-100 mg i/m for 12-
14 days or PRID/CIDR/TRIU-B/ Ear implants for 10-12 days.
xii. Urovagina: The condition is also known as vesicovaginal reflex or urine pooling
refers to presence of urine in the cranial vagina and possibly in the uterus.
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Urovagina causes vaginitis, cervicitis and endometritis. The diagnosis of urovagina

can be done by speculum examination at the time of estrus to detect the presence of
urine in the vagina. The condition can be corrected by vaginoplasty and more
correctly termed as caudal allocation of the transverse folds as surgical intervention
is in the vestibule region.
xiii. Pneumo-vagina: It is accumulation of air in the vagina. The condition can be
treated by Caslick‟s operation.
3) Managemental factors:
i. Improper heat detection: Bull parading is advised twice a day in the herd. In case of
few animals at door step, watch the animals closely thrice a day for heat symptoms
and record the dates of heat which help in expectancy of next heat.
ii. Environmental stress: Elevated temperature due to persistent fever or high
environmental heat and humidity may lead to the early embryonic death. Rest
before and after AI is advised because elevated cortisol interferes with LH
secretion thereby interfering with ovulation. Also, beating of animal before and
after AI is contraindicated. Avoid journey before and after AI.
iii. Nutritional deficiencies: Deficiency of wide range of specific nutrients has been
observed in poor reproductive performance. Particularly, Vitamin E and Selenium
are reported to cause early embryonic death. Feeding of estrogenic forages to the
cows and buffaloes also affects the embryonic survival. Feed the mineral mixture
regularly @ 50-100g. Bring the animal to positive nutritive energy balance.
iv. Improper timing of AI: Adopt AM-PM method for AI. If animal is observed in heat
in morning, then AI is advised in the morning and vice-versa.
v. Untrained inseminator/faulty AI: The AI should always be allowed by a trained
inseminator. The site of deposition of semen is body of uterus.
vi. Unhygienic conditions at the time of AI: Contamination of sheath with dung
introduces infection directly into the uterus. Therefore, strict hygienic measures
have to be adopted.
vii. Improper thawing of straw: Thawing of straw should be done in water bath at a
temperature of 35-370C for 20-30 seconds. High or low thawing temperature
interferes spermatozoal viability.

EARLY EMBRYONIC MORTALITY

Embryonic mortality denotes the death of fertilized ova and embryos up to the end of
implantation. About 25 to 40% of embryos are normally lost in farm species. It is also noted in
large litters of swine and during multiple pregnancies in cattle and sheep. Mortality is more
common during the early than the late embryonic period. Early embryonic mortality should be
regarded as a normal process of eliminating unfit genotypes in each generation, particularly in
large litters of swine and multiple pregnancies in cattle and sheep. In the past, it was believed that
the bovine conceptus is resorbed but transrectal ultrasound examination has demonstrated that the
conceptus and its breakdown products apparently are eliminated by expulsion through the cervix,
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which either goes unnoticed or appears as a vulval discharge of clear mucus. Embryonic mortality
after natural breeding or artificial insemination accounts for the majority of reproductive failures
in the cattle, with a mortality rate of up to 40% of all fertilized eggs. In cattle, most embryonic
deaths occur between days 8 and 16 during hatching of the blastocyst and implantation without
affecting cycle lengths. Since, most embryos die between days 9 and 15 infertile ewes may
experience normal as well as prolonged cycles.

Cattle

Most of the embryonic loss in cattle occurs between days 8 and 16 after insemination. The
timing of insemination is important as insemination too late in the estrous period leads to ovum
ageing and embryonic death. Artificial insemination during pregnancy will induce loss, either
through mechanical trauma to fetal membranes or the introduction of infection. Nutritional causes
such as B-carotene, selenium, phosphorus and copper deficiencies have been implicated in
embryonic loss. High intakes of crude protein, in particular rumen – degradable protein have been
associated with reduced fertility. This is said to be due to the toxic effects of blood urea or
ammonia on the embryo. Stress e.g. heat stress, has also been shown to result in embryonic loss.
High lactation yield is negatively correlated with fertility and this could be considered a metabolic
stress.

Horses

The commonest cause of embryonic loss in mares is twin conceptions as competition for
placental space usually results in one fetus growing more slowly than the other and the smaller
fetus, with a smaller placenta, dies. Death of one fetus often results in the loss of the second.
Other intrinsic factors which are thought to be related to embryonic loss in the mare include
oviductal secretions, embryonic vesicle mobility and uterine environment. Since, the mare‟s
embryo is at a more advanced stage whilst still in the uterine tube, the environment may be
relatively more important in this species than others. In addition the embryonic vesicle remains
free in the lumen of the uterine horn much longer in the mare than in other species and the degree
of mobility of this vesicle is thought to be important in maternal recognition of pregnancy. Greater
mobility enhances the suppression of luteolysis and results in higher levels of progesterone. As
regards the uterine environment, recurrent endometritis and post-service infection lead to
perivascular fibrosis, and this is a common cause of embryonic and fetal death between 40 and 90
days of gestation. Increased maternal age has also been associated with increased embryonic loss.
Other factors such as lactational stress, transport stress and service at the foal heat also result in
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higher embryonic death rates. Nutritional stress, in the form of restricted energy intake, does
increase embryonic loss.

Sheep

Nutrition, specifically energy level, is known to affect embryonic survival in sheep in a

complex manner. Low body condition at mating is detrimental to embryonic survival, irrespective
of post mating nutrition. However, in ewes that lose weight post-service, embryonic mortality is
increased. Prolonged, moderate under nutrition has more effect on ewe lambs than adult ewes.
Nutritional energy may exert its effect via peripheral blood progesterone between food intake and
progesterone levels. Other nutrients important in embryo survival are vitamin E and selenium.
Certain plants, such as kale and Veratrum californicum, will cause embryonic death. Veratrum
californicum is also a teratogenic agent. In breeds with very high ovulation rates the embryonic
death rate rises proportionately, but this is probably due to limitations of uterine space. High
environmental temperature, particularly in the first week after mating, has been shown
experimentally to increase the embryonic death rate dramatically. This could be important in
climatic heat waves. However, if there is diurnal variation, as would occur naturally, the loss is
much lower. Physiological stress, such as that produced by overcrowding or handling of sheep,
also increases embryonic loss. This may be due to excess secretion of corticosteroid by the
adrenals.

Goats

Goats are particularly susceptible to non-infectious fetal loss, and this is particularly true of the
Angora breed. Losses are also common in poorly fed animals of any breed. Another reported
cause of fetal loss is dosing with anthelmintics such as carbon tetrachloride and phenothiazine.

Pigs

Ovulation rate is not usually a limiting factor in productivity in the pig but, in general, as
ovulation rates increase, the embryo survival rate decreases. This can be demonstrated in gilts,
where the ovulation rate can be artificially increased but embryo survival rate decreases. Even if
early embryonic death does not occur with high ovulation rates, a problem may arise later in
pregnancy with competition for uterine space. It has been suggested that a higher fetal death rate
exists when there are more than five fetuses per horn, with those embryos in the middle of the
horn being smaller.
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Apart from the above intrinsic factors, extrinsic factors such as nutrition and stress play an
important part in embryonic loss in the pig. For example, it is well documented that high
energy levels after service result in reduced embryo survival. Stress, associated with
extremes of temperature, or certain management systems such as sow stalls or tethers, is
also known to result in increased embryo mortality. Other husbandry policies such as
lactation length, also affect embryonic death rates, and lactation lengths of less than 3
weeks produce a marked rise in embryonic mortality presumably due to a poor uterine
environment.

Sequel to Embryonic or Fetal Death

Following early embryonic death the embryonic tissue are usually resorbed, and the animal
returns to estrus if there is no other conceptus in the uterus. If death occurs before the maternal
recognition of pregnancy the estrous cycle is not prolonged. If it occurs after recognition has taken
place, the estrous cycle will be prolonged. If death of the embryo is due to an infection then, even
though the embryonic material may be absorbed, a pyometra may follow. In cattle this condition is
characterized by persistence of the corpus luteum, closed cervix and pus accumulation in the
uterine body and horns. It is a particular characteristic of infection with Tritrichomonas fetus. If
fetal death occurs after ossification of the bones has begun, complete resorption of fetal material
cannot take place, instead, fetal mummification occurs.

Causes for early embryonic death

Embryonic mortality can be due to maternal factors, embryonic factors, or to embryonic-

maternal interactions. Maternal failure tends to affect an entire litter, resulting in complete loss of
pregnancy. In contrast, embryonic failure affects embryos individually, often leaving others in
litter unharmed. In other cases the maternal environment may be insufficient, allowing the support
of only a few embryos.

1. Endocrine Factors
Accelerated or delayed transport of the egg, as a result of estrogen – progesterone
imbalance, leads to preimplantation death. An abnormally undersized conceptus might not
be able to counteract the uterine luteolytic effect, with consequent regression of the CL and
termination of pregnancy. In swine, as stated previously, at least four living blastocysts are
needed by day 10 of pregnancy to counteract the uterine luteolytic effects. A critical
period of embryonic survival is the late blastocyst stage. Normally, the developing CL
secretes progesterone, which acts on the female tract in close synchrony with the
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development of the embryos. Insufficient progesterone at the time of implantation leads to

establishment of pregnancy.

2. Lactation
Embryonic mortality occurs during lactation in cattle, sheep, and horses and is
characterized by prolonged estrous cycles after breeding. Mating of mares at foal heat
leads to early embryonic mortality, which has been attributed to reduced effectiveness of
uterine defence mechanisms, stress of lactation, and incomplete regeneration of the
endometrium. Sows bred after weaning at 7 days of lactation suffer high embryonic losses
between days 9 and 20 of pregnancy.

3. Nutrition of the Dam

Caloric intake and specific nutritional deficiencies affect ovulation rate and
fertilization rate, as well as cause embryonic death. Also extremes in the level of feeding
are detrimental to embryo survival, so too are extremes in the supply of specific dietary
nutrients. In dairy cows, high intakes of rumen degradable protein may lead to embryonic
mortality. This effect may be mediated through a reduction in the pH of the uterine
environment during the luteal phases of the cycle in which the embryo must grow. In
swine, high caloric intake or continuous unlimited feeding increases ovulation rate, thereby
increasing the incidence of embryonic mortality before implantation. However, following
implantation, unlimited feeding decreases fetal death. In sheep, full feeding before
breeding also increases ovulation rate as well as embryonic mortality. Poor body condition
of ewes at mating increases the incidence of embryonic mortality, whereas moderate feed
restriction from day 20 to 100 of pregnancy is less likely to reduce lambing percentages.
Under nutrition affects twin ovulators more than single ovulators because both embryos
are lost in twin ovulators, while a single embryo survives in case of single ovulators. Thus,
more twin than single ovulating ewes are barren. No resorption occurs if mares are
maintained on an adequate plane of nutrition until 35 days after service.

4. Age of the Dam

A higher incidence of embryonic mortality is observed in gilts and in sows after the
fifth gestation. In the ewe, the incidence of late embryonic loss is higher in ewe lambs and
ewes over 6 years than it is in mature ewes, which is due to factors associated with the
embryo rather than the uterine environment.
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5. Overcrowding in Utero

Because the degree of placental development is primarily influenced by the

availability of space and vascular supply within the uterus, increasing the number of
implantations decreases the vascular supply to each site and restricts placental
development. This results in a high embryonic and fetal mortality rate and probably
explains the higher incidence of embryonic mortality in cattle and sheep following twin
rather than single ovulation. It should be noted that uterine capacity does not limit the
ability of the cow and ewe to carry twins, provided they are located in separate uterine
horns. In cattle, embryo transfer experiments have shown a higher embryonic mortality
rate in recipients which received two embryos in a single uterine horn. This loss may be
due to overcrowding and intrauterine competition for nutrients. In cattle and sheep with
multiple ovulations, the number of embryos surviving is reduced to a fairly constant
number within the first 3 or 4 weeks of pregnancy, which implies that embryonic loss
increases as the number of eggs shed increases. Mortality does not seem to be due to a
deficiency of progesterone. In prolific breeds of sheep, late embryonic deaths occur in
ewes with more than five ovulations. Transuterine migration of embryos is of importance
for equal distribution of embryos in the two horns of the uterus in polytocous species such
as swine. In its absence, there is a high incidence of embryonic mortality in swine.

6. Thermal Stress

Embryonic mortality increases in a number of species following exposure of the

mother to elevated ambient temperatures; especially in tropical areas the effects of thermal
stress on the early embryo are not apparent until the later stage of its development.
Fertilized eggs of sheep and cattle, when subjected to high temperatures either in vitro or
in vivo are damaged but continue to develop, only to die during the critical stages of
implantation. Reduced fertility of summer heat-stressed dairy cows may result from
decreased viability and developmental capacity of 6 day old to 8 day old embryos and may
account for the well-documented seasonal reduction in the efficiency of artificial
insemination during summer. Heat stress between days 8 and 17 of pregnancy may also
alter the uterine environment as well as growth and secretory activity of the conceptus.
Apparently heat stress antagonizes the inhibitory effects of the embryo on the uterine
secretion of PGF2α. Pig embryo is most susceptible to heat stress before day 18 of
pregnancy particularly during implantation.
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7. Semen

A portion of all embryonic mortality is attributable to the male and the mating system.
Genetic factors that are transmitted by the male to the embryo may be inherited, may arise
from testicular tissue, or may occur in spermatozoa after they are released from the testis. In
swine, semen stored for 3 days before insemination produced zygotes much more susceptible
to early embryonic death, presumably owing to the reduced DNA content in aged
spermatozoa.

8. Incompatibility

The inherited genotype of the male may include a variety of genetic factors that
lead to incompatibility and early embryonic loss. There may be incompatibility between
spermatozoa and mother, between spermatozoa and egg, or between zygote and mother.
Immunologic incompatibilities may block fertilization or cause embryonic, fetal, or
neonatal mortality. In cattle, homozygosity for certain blood groups and certain substances
related to transferring and J-antigen in sera are associated with increased embryonic loss as
well as decreased fertilization rate.

DIAGNOSIS AND TREATMENT

Diagnosis

Based on reproductive history and clinical examination

Treatment

Specific treatments for conditions like delayed ovulation, endometritis may be carried out. Since
most of the cases do not reveal any specific condition the following guidelines may be adopted.

 Bring the animal into positive energy balance.

 Do AI twice at each estrus preferably at 12 or 24 h interval.
 Check the semen quality - use only high quality semen
 Clitorial stimulation for at least three seconds at AI.
 Administration of 1000-1500 IU of hCG/ 50mg GnRH (10 mg of GnRH analogue) at the
time of AI.
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 Administration of 500 mg of depot progesterone on the 5th day of AI.

 Skipping of AI, administration of PGF2α after 9-10 days and fixed time AI twice at 72 and
96 h.
 Skipping of AI and intrauterine infusion of 2 ml of Lugol‟s solution diluted in 8 ml of
sterile saline.
 Flushing of the uterus with normal saline - under moderate pressure as being done in
embryo transfer (to remove cellular debris and also mild block in the uterine tubes).

FAILURE OF FERTILIZATION

Fertilization failure may result from death of the egg before sperm entry, structural and
functional abnormality in the egg or sperm, physical barriers in the female genital tract preventing
gamete transport to the site of fertilization, or ovulatory failure.

CAUSES OF FERTILIZATION FAILURE

Abnormal egg

FERTILIZATION
Gamete transport Structural barriers
FAILURE

Abnormal sperm

CAUSES:

Abnormal eggs
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 Several types of morphologic and functional abnormalities have been observed in

unfertilized eggs, e.g., giant egg, oval shaped egg, lentil-shaped egg, and ruptured zone
pellucida.
 Failure to undergo fertilization and normal embryonic development may be due to inherent
abnormalities of the egg or to environmental factors.
 In sheep, some of the conception failures at the beginning of the breeding season are
associated with a high incidence of abnormal ova.

Abnormal Sperms

The physiologic significance of abnormal sperm in relation to fertilization failure has

not been studied in animals other than cattle. Certain forms of male infertility are related to
structural defects of the DNA protein complex. Sperm aging and injury may cause-

o Alterations in the acrosomal cap that may prevent defective spermatozoa from
fertilizing the egg. In bull, ram, and boar, a good correlation exists between fertility
and acrosomal integrity.
o Leakage of vital intracellular constituents such as cyclic AMP or the formation of
lipid peroxides from sperm membrane when sperm are stored under anaerobic
conditions.
o A gradual decrease in the fertilizing capacity of aging of spermatozoa in the female
genital tract.

Structural Barriers to Fertilization

Congenital or acquired defects of the female genital tract interfere with transport of
the sperm and/or the ovum to the site of fertilization-

 Congenital defects are the result of arrested development of the different segments
of the Mullerian ducts or of an incomplete fusion of these ducts caudally. A classic
congenital anomaly associated with the gene for white coat color is “white heifer
disease” in cattle, in which the prenatal, development of the Mullerian ducts is
arrested, and the vaginal canal is obstructed by the presence of an abnormally
developed hymen. It can be differentiated from the freemartin syndrome by the
presence of normal ovaries, vulva, and labia.
 Common anatomic abnormalities are adhesions of the infundibulum to the ovary or
uterine horns; this interferes with the pick-up of the egg or causes a mechanical
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obstruction of one part of the reproductive duct system. Bilateral or unilateral

missing segments of the reproductive tract also cause anatomic sterility.

Phytoestrogens

 Reproductive failure occurs more in sheep than in cattle grazing on plants that contain
compounds with estrogenic activity, e.g. subterranean clover and red clover.
 The estrogenic activity is due to plant isoflavones and related substance with hydroxyl
groups. Cows and ewes fed estrogenic forage may suffer impaired ovarian function, often
accompanied by reduced conception rates and increased embryonic loss. In cows, clinical
signs resemble those associated with cystic ovaries.
 The infertility is temporary, normally resolving within one month after removal from the
estrogenic feed. Ewes grazed on estrogenic pastures around the time of joining, shed fewer
ova and have a reduced chance of conception.
 Fertility is improved within 3 weeks, after the ewes are moved into non-estrogenic
pastures. The pathologic changes in temporary infertility are due to actions of estrogen on
the hypophyseal – ovarian axis and on sperm transport.
 Ewes grazed for several seasons on estrogenic pastures mate and ovulate, but fertilization
rate is depressed as a result of failure of sperm transport caused by severe changes
occurring in the cervix.

TRANSMISSIBLE VENEREAL TUMOR (TVT) IN CANINES

Transmissible venereal tumor (TVT), also known as infectious sarcoma, venereal

granuloma, transmissible lymphosarcoma or Sticker tumor is a benign reticuloendothelial tumor of
the dog that mainly affects the external genitalia and occasionally the internal genitalia. As it is
usually transmitted during coitus, it mainly occurs in young, sexually mature animals. It is
transplanted during coitus with intact viable cells across major histocompatibility complex (MHC)
barriers within the same species and even to other members of the canine family, such as foxes,
coyotes and jackals. Laboratory transplantation of TVT from one dog to other using viable cells is
also possible. TVT cells contain an abnormal number of chromosomes ranging from 57 to 64 and
averaging 59, in contrast to the normal 78 of the species. Metastasis of TVT is uncommon, only
occurring in puppies and immuno-compromised dogs. The uniqueness of TVT lies in the fact that
this is the only proven example of a naturally occurring tumor that is transmitted as an allograft by
cell transplantation, and the tumor becomes autonomous from the original host. In other words,
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the tumor behaves like a parasite. Growth of tumour occurs 15 to 60 days after implantation. This
kind of tumor developed only in the dog, probably because during coitus there is extensive
abrasive abrasions and bleeding of the penile mucosa and vagina, making transplantations of the
tumor easy.

TVT has continued to be a serious problem around the world occurring at same
frequencies in both male and female dogs. It is commonly observed in dogs that are in close
contact with one another, or in stray and wild dogs that exhibit unrestrained sexual activity. In
India TVT is known to be the most frequently reported tumor in dogs ranging from 23-43 % of the
total number of tumors in canine population. Metastasis may occur in less than 5-17% of cases.
Uncontrolled sexual behavior and a large stray dog population appear to be one reason for such a
high incidence of TVT.

GROSS AND MICROSCOPIC CHARACTERISTICS

The transmissible venereal tumor affects the vagina and external genitalia of the bitch and
the penis of the dog. Transmission of the tumor occurs at the coitus when infected cells „seed‟ the
genital mucosa of the recipient. Autotransmission of the nasal and oral mucosa may occur by
licking of the tumor. The lesions are often friable, multilobulated and may be single and multiple,
generally reach their maximum size after 5-7 weeks and may regress spontaneously within 6
weeks. Small pink to red, 1 mm to 3 mm diameter nodules can be observed 2 or 3 weeks after
transplantation. Multiple nodules then fuse together forming larger, red, hemorrhagic, cauliflower-
like, friable masses of 5 to 7 cm in diameter which then progress deeper into the mucosa as
multilobular subcutaneous lesions with diameters that can exceed 10-15 cm. Tumours bleed easily
and while becoming larger, normally ulcerate and become contaminated. Exfoliative cytology
shows discrete cells that are round to oval, with moderately abundant pale blue cytoplasm, an
eccentrically located nucleus, with occasional binucleation and mitotic figures. Single or multiple
nucleoli are often observed. The most characteristic feature of TVT cells is the presence of
numerous discrete clear cytoplasmic vacuoles.
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Transmissible Venereal Tumor in bitch. Transmissible Venereal Tumor in dog

TVT Cells-Leishmans Stain (100X)

DIAGNOSIS
Clinical signs vary according to the localization of the tumors. Dogs with genital
localization have a hemorrhagic discharge. In males, lesions usually localize cranially on the glans
penis, on preputial mucosa or on the bulbus glandis. Tumoral masses often protrude from the
prepuce and phimosis can be a complication. The discharge can be confused with urethritis,
cystitis, or prostatitis. The involvement of regional lymph nodes is frequent in males with large
tumors. In bitches the tumors are of similar gross appearance as in male dogs and can be localized
in the vestibule and/or caudal vagina, protruding from the vulva and frequently causing a
deformation of the perineal region. Only very rarely, however, do they interfere with micturition.
A considerable hemorrhagic vulvar discharge may occur and can cause anemia if it persists. The
discharge can attract males and the condition of the bitch can be mistaken for estrus by the
owners. Infrequently, TVTs can localize in the uterus. In cases with extra genital localization of
the TVT, clinical diagnosis is usually more difficult because TVTs cause a variety of signs
depending on the anatomical localization of the tumor, e.g., sneezing, epistaxis, epiphora, and
tooth loss, exophthalmos, skin bumps, facial or oral deformation along with regional lymph node
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enlargement. Exfoliative vaginal cytology has been one means of diagnosing TVT in the bitch.
Definitive diagnosis is based on physical examination and cytological findings typical of TVT in
exfoliated cells obtained by swabs, fine needle aspirations or imprints of the tumors. There is
marked aberrations in the numbers and morphology of the chromosomes of the constituent cells of
TVT.

TREATMENT
Several treatments including surgery, radiotherapy, immunotherapy, and chemotherapy
have been applied for TVT. Surgery has been used extensively for the treatment of small,
localized TVTs, although the recurrence rate can be as high as 50 - 68% in cases of large invasive
tumors. Contamination of the surgical site with TVT cells is also a source of recurrence. Methods
to prevent recurrence subsequent to surgery include excision along with cauterization,
electrosurgical or cryosurgical excision or chemotherapy subsequent to surgical excision.

Chemotherapy has been shown to be the most effective and practical therapy, with vincristine
sulfate being the most frequently used drug Vincristine, is administered weekly at a dose of 0.5 to
0.7 mg/m2 of body surface area or 0.025 mg/kg, i/v. The involution of the lesions is gradual,
although it is particularly noticeable and significant at the beginning of the treatment. Complete
remission usually takes 2 to 8 injections and occurs in more than 90% of the treated cases. A cure
rate approaching 100% is achieved in cases treated in the initial stages of progression, especially
in cases of less than 1 year duration, and independent of the presence or not of metastases. In cases
of longer duration, longer periods of therapy are required, and the cure rate is lower Cytostatic
agents, such as vincristine, can cause myelosuppression and gastrointestinal effects resulting in
leucopenia and vomiting in 5 to 7% of the patients. Paresis has also been described as a side effect
due to peripheral neuropathy. A complete white blood cell count is, therefore, recommended prior
to each administration. When the white blood cell count is below 4,000 mm3 further
administration should be delayed 3 to 4 days and the dose of vincristine can be reduced to 25% of
the initial dose. The most frequent complication of vincristine treatment is the occurrence of local
tissue lesions caused by extravasation of the drug during i/v administration resulting in the
development of necrotic lesions with crusts. Cyclophosphamide @ 5 mg/kg PO for 10 days as a
single drug therapy or in combination with prednisolone @ 3 mg/kg, for 5 days; vinblastine @ 0.1
mg/kg IV for 4-6 weeks; methotrexate @ 0.1 mg/kg PO every other day can be given alone or in
combination for treatment of TVT. There is no advantage of combination therapy over vincristine
administration alone. In cases that fail to resolve with chemotherapy, electro-cauterization, cryo-
cauterization or radiotherapy has found to yield good results.
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IMMUNE-MODULATION FOR ENHANCEMENT OF FECUNDITY

Immunomodulaton in reproductive disorders of livestock is a new area of

immunotherapeutics. It is mainly used to treat inflammatory conditions of the reproductive
disorders such as endometritis, metritis, vaginitis in which the immunity barrier of the host is
breached. Immunomodulaton is also done using plant preparations. The normal uterine defence
mechanism is brought about by:
 The anatomical barriers which include vulva, vestibule and cervix.
 The physical barrier which includes the cervical-vaginal mucous.
 The contractions of circular and longitudinal uterine muscles that help to propel the
microbes outside.
 Chemical barrier which consists epithelial and stromal cells which secrete soluble factors
and cause cell to cell adhesions.
 Immunological barrier constituted by the cellular immunity comprising of
polymorphonuclear inflammatory cells (PMNs), lymphocytes and humoral immunity by
antibodies.

An ideal immunomodulator should meet the following criteria:

 It should be non-toxic for animals even at high doses.

 It should not have teratogenic, carcinogenic or any other side effect.
 It should have a short withdrawal period with low tissue residues. It should not be secreted
in milk or meat.
 It should stimulate both specific as well as paraspecific immune responses in the body.
 It should act as adjuvant when given with vaccines.
 The immunomodulator or its breakdown products should be either inactive or readily
biodegradable in the environment.

UTERINE CELLULAR AND HUMORAL IMMUNITY

Uterine Cellular Immunity

 Polymorphonuclear inflammatory cells (PMNs), blood monocytes and tissue macrophages

are regarded as „professional phagocytes‟ in the cellular defences against pathogenic
microorganisms.
 Phagocytosis involves chemotaxis, adherence and attachment of leucocytes to cell surface
antigens presented by the organism before it is ingested by the phagocyte and finally
digested.

Uterine Humoral Immunity

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 Immunoglobulin concentrations in uterine secretions reflect both the extent of the

endometrial inflammatory process in the face of microbial challenge.
 Immunoglobulins have been found in bovine uterine secretions and their protective role
against pathogens has been widely reported.

INFLUENCE OF STEROID HORMONES ON UTERINE IMMUNE RESPONSES

 The cyclical pattern of steroid hormone concentrations, characteristic for different stages
of estrous cycle, regulates potential pathogenicity of micro-organisms that contaminate the
uterus postpartum.
 Steroid hormones influence functional activity of leucocytes migrating into uterus.

 During estrogen phase of the ovarian cycle there is increased mucus production,
and identified PMN activity;
 During the luteal phase, there is reduced endometrial epithelial permeability to
bacteria that delays leukocyte stimulation, and an absence of detoxifying agents in
uterine secretions.

 Disruptions of these mechanisms allow opportunist pathogens, mostly microorganisms

found in posterior gastrointestinal tract and around perineal area.
 Inflammation of endometrium can occur following:

 coitus,
 artificial insemination (AI),
 More commonly in cattle after parturition and in majority of cases 1-4 weeks after
calving,

 Micro-organisms contaminate the uterine lumen but self-cure usually occurs within 6
weeks postpartum. In those cows unable to eliminate infection, endometritis may develop
subsequently. Endometritis causes a significant delay in calving to conception intervals.
 Intrauterine oxygen reductase potential (Eh) can be used to find the degree of bacterial
infection as more Eh potential indicates infection. Alkaline pH of uterus indicates uterine
infection. Peripheral blood haptoglobin, which is an acute phase protein synthesized in the
liver in repose to tissue damage has been used as a marker for endometritis; its major
function is to bind free haemoglobin and protect the host from the oxidative activity of
haemoglobin. Another circulating acute phase protein, α1-acid glycoprotein is also
screened for detecting endometritis.

USE OF IMMUNOMODULATORS IN TREATMENT

 Plenty of research associated with immunomodulators of uterine defence mechanism and

their application as treatments for metritis or endometritis has been carried out in mares.

 Commonly used as immunomodulators are:

 Prostaglandins
 E.Coli Lipopolysaccharides
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 Derivatized Polysaccharides
 Oyster Glycogen
 Leukotriene B4

Other Agents

Other agents commonly used as immunomodulators are - Plant Preparations

HORMONES

Prostaglandin

Prostaglandin F2α is a naturally occurring luteolytic agent in animals. Oxytocin

stimulates uterine secretion of PGF2α which in turn, stimulates oxytocin release from the
corpus luteum; this luteal oxytocin positively stimulates endometrial secretion of PGF2α.
As a result, peripheral blood concentrations of progesterone progressively fall from day 16
of the estrous cycle until estrus, when they are undetectable; conversely, estradiol
concentrations rise to reach a peak when the animal shows standing heat. Exogenous
prostaglandin therapy administered to cattle presenting a functional corpus luteum will
induce luteolysis and bring the animal into heat, thereby removing the suppressive affect of
progesterone on the uterine defence mechanism or, alternatively, stimulate it through
estrogen causing myometrial contraction which expels debris and micro-organisms that
contaminate the uterine lumen after calving. Moreover, PGF2α may have stimulatory effect
on the phagocytic activity of uterine leucocytes. Hence, the luteolytic action of PGF 2α has
been used to treat endometritis in cows where a functional corpus luteum is present.

E.coli Lipopolysaccharides

 E. coli lipopolyaccharides (LPS) are thought to act as a chemo-attractant to PMNs. This

increase in numbers of PMNs in the endometrium may help to resolve endometritis in both
cows and mares. A single intrauterine infusion of 100 μg E. coli LPS at estrus in repeat
breeding cows clears the bacterial infection from the uterine lumen within one estrous
cycle. Similar results have been described following intrauterine administration of LPS in
ewes. When E. coli endotoxin administered similarly to cows 5 days after calving at rates
of 5 g/kg body-weight, the toxin is absorbed with associated transient clinical signs
causing enhancement in immune response.
 The contaminating micro-organisms associated with uterine infection postpartum may
themselves secrete endotoxins detectable in peripheral blood, and may interfere with
ovarian function characterized by short cycles in cows. Variation between species in
endotoxin absorption from uterus postpartum may be associated with differences in
placentation and the extent of trophoblast invasion of the endometrium. In species, where
there is little clinical or physiological evidence of endotoxin absorption, an intact
endometrium may act as a cytological barrier to totally or partially exclude the toxin.

Derivatized Polysaccharides

 They are generally high molecular weight dextrans.

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 Their action is complex possibly by T or B cell mitogenicity e.g. oyster glycogen.

Oyster Glycogen

 Intrauterine administration of oyster glycogen (OG) caused PMN migration, up to

90% of all cells identified in uterine secretions being neutrophils.
 Measurable immunoglobin (IgG) concentrations were found in uterine secretions
following administration.

Leukotriene B4

 They are generally glycoproteins of molecular weight around 20 kDa.

 They up regulate immune response. They are effective chemo-attractants, stimulating
preferential migration of PMNs into the lumen of the bovine uterus.
 A single intrauterine treatment of a 30 mmo1/l solution increased the intrauterine
leukocyte count 5-10 times within 24h.
 Granulocyte-macrophage colony stimulating factor (GM-CSF; lymphokines) are highly
effective chemo-attractants in mares, but their activity in cattle has not been studied.

OTHER AGENTS USED AS IMMUNOMODULATORS

Plants Used in Disorders

 Uterine disorders
o A wide variety of medicinal plants and their preparations are found to be useful in
treatment of reproductive ailments.
o They include Abroma augausta, Aristolochia bracteata, Datura alba, Mytrus
communis, Salvadora species, Saraca indica, and ViI species.
 Anestrum
o Anestrum in cows/buffaloes can be overcome by feeding fenugreek powder.
Feeding of bamboo leaves brings cattle and buffaloes into regular heat. Feeding of
leaves of jute plant (about 2-2.5 kg) brings animal into heat.
o Feeding leaves of Mann tree (approx:15-20 kg) can overcome anestrus condition.
o A mixture of black pepper (10 grains) and Vanghuchi (20-25 gm) is given twice a
day at interval of 6-8 hrs for 1-2 days for treatment of anestrus.
 Decreased Conception Rate
o Feeding 200 gm germinated Bengal gram (Cicer arietnum) soaked overnight to the
animal continuously for one week.
o Along with this pounded leaves and unopened fruit of Yanai (Pedalium murex)
may be given once a day for three days without adding water.
 Retained Placenta
o Bamboo leaves and bark are boiled with paddy husk and fed to cows for expulsion
of placenta.
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o Ficus bengalensis is used for treatment of retained placenta. Leaves and twigs of
ber (Zyzphus mauritiana) are collected and burnt. The ash is given to the animal
with water to induce the placenta to drop.
o About 250 g of leaves of jingara used for retention of placenta showed 60% success
rate.
o To prevent abortion farmers feed piece of stem of banana (Musa
paradisiacal).After conception, buffalo is fed 10-15 kg; cow is fed 5-10 kg pieces
of stem of banana. It is fed for five times over a period of 2-3 days. It helps to
reduce internal heat and improves health. This practice has been in use for the last
30-40 years.
 Uterine Infection
o The immunomodulatory property of Aristolochia indica (Ischamur) can be proved
an aid in preventing the uterine infection by augmenting local immune system.
Approximately 100 gm of root or bark of Convolvus micrphyllus (roots) powdered
and mixed with 300 ml of water and boiled. This concoction is filtered and then
cooled. This is given to affected animals once a day for 3 days.
 Equiman
o This is an immunomodulating phytopreparation having its affect on bovine immune
cells. Equimen is a fixed combination of Echinaces purpurea, Thuja occidentalis
and elemental phosphorous in different concentration. The preparation reduces the
spontaneously generated reactive oxygen species (ROS) by neutrophils.
o Phytotherapy has been followed in the treatment of animals from thousands of
years since ancient time. Plant based drugs (natural drugs) may be used directly i.e.
they may be collected, dried and used as a therapeutic agents (crude drugs) or their
active principles, separated by various chemical process which are employed as
medicines.

Increasing fecundity: Immunization against steroid hormones to increase lambing

percentages

Immunization of the ewes against their own oestrogenic steroid hormones is a new method
of increasing lambing percentages of sheep due to the increase in the ovulation rate. This
technique offers the potential of an immediate gain in ovulation rate and lambing percentages.
Active immunization is considered likely to be more practical than a passive system. Ewes are
vaccinated against their own estrogenic steroid hormones and this has the effect of upsetting the
system which regulates the number of eggs shed. The application of immunization increases
lambing percentage by between 20 to 30% in breeds like Corriedales that have currently a low
twinning percentage. Obviously the increase in fecundity requires good management and food
availability, so the application of these methods is limited to farms in which this requirement can
be achieved.

Immunization against Inhibin

Inhibin is secreted by the gonads of mammals and it plays an important role in the control
of FSH release. Inhibin is a non-steroidal glycoprotein hormone of gonadal origin with major
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action as negative feedback control of the production of Follicle Stimulating Hormone (FSH) by
anterior pituitary gland which in turn modulates male and female reproductive functions. Its
physiological role has led to the development of inhibin based immunogens for fertility
enhancement in farm animals. It is envisaged that a reduction of endogenous inhibin secretion
would increase FSH concentrations and thus, offers a potential for increasing the number of
ovulatory follicles in the ovary. Immunization against inhibin has been reported to be a useful
method for inducing multiple ovulations in farm animals. Inhibins play important roles in the
regulation of fertility based on their dual inhibitory action on the process of folliculogenesis in the
ovary and FSH secretion by the pituitary. Inhibins are also recognized as paracrine ovarian and
testicular regulators and have multiple paracrine effects in the utero-placental unit, representing a
promising marker for male and female infertility, gynecological and gestational diseases. Inhibin
essentially suppresses FSH; immunisation against inhibin is being examined as a way of
increasing ovulation rates in females and generally increasing fertility in males.

Immunization against Gonadotrophin Releasing HormoneAdd for external exam::